Mutant viral capsid libraries and related systems and methods

ABSTRACT

Provided are mutant viral capsid cell libraries, individual cells of such libraries, systems, vectors, and methods for generating the cell libraries, and methods of use thereof to screen for mutant viral capsids with desired characteristics.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/387,177, filed Dec. 23, 2015, the full disclosure of which isherein incorporated by reference.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is AVBI_008_01US_ST25.txt. The text file is 2 KB,was created on Dec. 21, 2016, and is being submitted electronically viaEFS-Web.

FIELD OF THE INVENTION

Embodiments of the present disclosure relate to mutant viral capsid celllibraries, particularly for mutant AAV capsids, individual cells of suchlibraries, systems and methods for generating the cell libraries, andmethods of use thereof to screen for mutant viral capsids with desiredcharacteristics.

BACKGROUND

A promising approach to treating and preventing genetic diseases anddisorders is delivery of therapeutic agents with a gene therapy vectorsuch as a viral vector. Illustrative examples of viral vectors suitablefor gene therapy include but are not limited to retroviral vectors,lentiviral vectors, adenovirus vectors, herpes virus vectors,alphaviruses vectors, and adeno-associated virus (AAV) vectors. AAV is a4.7 kb, single stranded DNA virus. Recombinant vectors based on AAV areassociated with excellent clinical safety, since wild-type AAV isnonpathogenic and has no etiologic association with any known diseases.In addition, AAV offers the capability for highly efficient genedelivery and sustained transgene expression in numerous tissues,including eye, muscle, lung, and brain. Furthermore, AAV has shownpromise in human clinical trials. One example is Leber's congenitalamaurosis in which patients treated with a therapeutic delivered by asingle subretinal administration of an rAAV vector have experiencedsustained clinical benefit from expression of the therapeutic agent formore than four years from the initial date of treatment.

Certain challenges that remain with regard to the design of viralvectors for use in gene therapy include optimizing viral cell tropismand reducing anti-viral or neutralizing host antibody responses. Forcertain viral vectors, such as AAV, cell tropism and neutralizingantibody responses result largely from the structure of the viral capsidprotein. Thus, there is a need in the art for improved tools to screenfor mutant viral capsids with desired properties. The present inventionaddresses these and other issues.

BRIEF SUMMARY OF THE INVENTION

Some embodiments of the present invention include an isolated cell,comprising a heterologous polynucleotide having a coding sequence thatencodes a non-naturally occurring mutant viral capsid, wherein thecoding sequence for the capsid is operably linked to a promoter on theheterologous polynucleotide, and wherein the heterologous polynucleotideis integrated into the genome of the cell. In one aspect, theheterologous polynucleotide is integrated into the genome of the cell ata pseudo attP site in the cellular genome. In one form, the isolatedcell does not encode more than one mutant viral capsid. In one form, theisolated cell comprises one but no more than one heterologouspolynucleotide having a coding sequence that encodes a non-naturallyoccurring mutant viral capsid. That is, an isolated cell of thisdisclosure preferably does not contain more than one integrated mutantviral capsid gene or coding sequence.

In one embodiment, the heterologous polynucleotide is integrated intothe genome of the isolated cell at a pseudo attP site in the cellulargenome and there are no other capsid-encoding heterologouspolynucleotides integrated into the genome of the cell, such that theisolated cell encodes a single structurally distinct mutant capsid.Accordingly, the mutant viral capsids produced by the isolated cell arestructurally identical. That is, the mutant capsids produced by theisolated cell do not comprise a mixture of two or more different mutantcapsids. In some forms of the invention, only a single heterologouspolynucleotide is integrated into the cell and the polynucleotide isintegrated into the cellular genome at a pseudo attP site. In a furtheraspect, the heterologous polynucleotide further comprises invertedterminal repeats (ITRs).

In some embodiments, the heterologous polynucleotide further comprises acoding sequence encoding a reporter protein, wherein the coding sequencefor the reporter protein is operably linked to a promoter. In anillustrative form of the heterologous polynucleotide, ITRs flank thesection of the polynucleotide (e.g., the expression cassette on thepolynucleotide) comprising the coding sequences for the mutant viralcapsid and reporter gene and the promoters to which each coding sequenceis operably linked. Furthermore, the heterologous polynucleotide canalso comprise a coding sequence encoding a drug-resistance gene, whereinthe coding sequence is operably linked to a promoter. Because it iscontained on the heterologous polynucleotide, the coding sequence forthe drug resistance gene is also integrated into the genome of the cell,and in more specific forms is integrated into a pseudo attP site of thecell. In exemplary forms of the invention, the coding sequence for thedrug resistance gene is not flanked by ITRs and is located outside theITRs that flank the coding sequences for the mutant viral capsid and thereporter protein.

Some embodiments of the present invention include isolated cells,comprising a heterologous polynucleotide that encodes anon-naturally-occurring mutant viral capsid which is operably linked toa promoter and is flanked by Inverted Terminal Repeats (ITRs), whereinthe heterologous polynucleotide is integrated into the genome of thecell. In some embodiments the heterologous polynucleotide is flanked byhybrid integrase-specific DNA attachment sites.

In some embodiments, the isolated cell is a eukaryotic cell. In someembodiments, the eukaryotic cell is a mammalian cell or an insect cell.In specific embodiments, the mammalian cell is a HEK-293 cell, HEK-293Tcell, or a HeLa cell. In some embodiments, the insect cell is selectedfrom SF9 cells, sf21 cells, S2 (Schneider 2) cells, BTI-TN-5B1-4 cells,and Tni cells.

In some embodiments, the mutant viral capsid is a mutant AAV capsid.

In certain embodiments, the hybrid integrase-specific DNA attachmentsites are attR and attL.

In some embodiments, the cell has only one integration event of aheterologous polynucleotide that encodes a non-naturally-occurringmutant viral capsid.

In particular embodiments, the cell comprises a heterologouspolynucleotide that encodes a reporter protein that is operably linkedto a promoter.

In some embodiments, the reporter protein is selected from greenfluorescent protein (GFP), yellow fluorescent protein (YFP), redfluorescent protein (GFP), mCherry, mRaspberry, mPlum, mTomato, dsRed,and luciferase.

In some embodiments, the cell comprises a heterologous polynucleotidethat encodes a drug-resistance gene which is operably linked to apromoter.

In some embodiments, the drug-resistance gene is selected from pac(puromycin), bsd (blasticidin), neo (G418), hygB (hygromycin B), and Shble (zeocin), Sh bla (gentamycin).

Accordingly, in some embodiments the heterologous polynucleotide canfurther encode a reporter protein and/or a drug-resistance gene. In someembodiments, the heterologous polynucleotide can comprise a codingsequence that encodes a reporter protein and a coding sequence thatencodes a drug resistance protein. Each coding sequence is preferablyoperably linked to a promoter.

In certain embodiments, the mutant viral capsid is a mutant AAV capsid,and the cell comprises (a) a rep-expressing polynucleotide that encodesone or more of Rep78, Rep68, Rep52, and/or Rep40 from AAV, and anAd-helper polynucleotide.

Also included are libraries of cells, comprising a plurality of isolatedcells described herein, wherein the non-naturally-occurring mutant viralcapsid of each cell in the library is distinct from the non-naturallyoccurring mutant viral capsid of substantially all of the other cells ofthe library. In some embodiments, the mutant viral capsid is a mutantAAV capsid. In some embodiments, substantially all of the plurality ofcells have only one integration event of a polynucleotide that encodes amutant viral capsid.

Some embodiments are directed to a library of cells, comprising aplurality of isolated cells described herein, wherein each cell in thelibrary encodes a single mutant viral capsid. In some forms,substantially all cells in the library, or at least 90% of the cells inthe library each encode a single mutant viral capsid. In other words,each cell in a library, substantially all cells in a library, or atleast 90% of the cells in the library comprise(s) no more than onemutant viral capsid gene per cell.

Some embodiments, include a library of cells (i.e., a mutant viralcapsid cell library) comprising a plurality of isolated cells asdescribed herein, wherein each cell in the library encodes a singlestructurally distinct mutant viral capsid, or wherein each cell ingreater than 50%, 60%, 70%, or 80%, or at least 90% of the cells in thelibrary encodes a single structurally distinct mutant viral capsid. Inpreferred aspects, the mutant viral capsid is a mutant AAV capsid.

In some aspects, the plurality of isolated cells in the library cancomprise, for example, greater than 10³, 10⁴, 10⁵, 10⁶ or greater than10⁷ cells. According to some aspects, the plurality of isolated cells isa plurality of isolated mammalian or insect cells. In more preferredaspects, the mutant viral capsid gene or coding sequence is integratedinto the genome of the isolated cell. In a more preferred aspect, thecapsid gene is integrated into a single site in the genome, and evenmore preferably the single site is a pseudo attP site in the cellulargenome.

Some embodiments include a mammalian cell library encoding or expressingmutant viral capsids, wherein each cell, or substantially all cells, orat least 90% of the cells in the library comprise(s) a differentheterologous polynucleotide that encodes a non□naturally-occurringmutant viral capsid, wherein the coding sequence for the non-naturallyoccurring mutant viral capsid is operably linked to a promoter, andwherein the heterologous polynucleotide is integrated into the genome ofthe cell using an exogenous or non-Rep integrase that integrates at anintegrase-specific DNA attachment site that is native to the cell. Inone aspect, the coding sequence for the mutant viral capsid and thepromoter to which it is linked are flanked by Inverted Terminal Repeats(ITRs). In one aspect, the integrase-specific DNA attachment site thatis native to the cell is a pseudo attP site in the cellular genome.Preferably, the heterologous polynucleotide is integrated into one sitein the genome, which is a pseudo attP site. In a further aspect, thenon-rep integrase is a serine recombinase or a phage integrase, such as,for example, any of those described herein. In a further aspect, themutant viral capsid is a mutant AAV capsid. The cell library ispreferably diverse. That is, the library preferably expresses, andenables the production of a plurality of distinct or unlike mutant viralcapsids, which ultimately assemble to form a plurality of distinctvirions whose capsids differ from one another.

Also included are cell culture devices, comprising an isolated cell or alibrary of cells as described herein.

Certain embodiments relate to systems for generating a mutant viralcapsid cell library, comprising a system for inserting a plasmid into anative DNA attachment site in the genome of a eukaryotic cell,comprising

(a) a vector encoding a mutant viral capsid which is operably linked toa promoter and flanked by Inverted Terminal Repeats (ITRs), andcomprising an integrase-specific DNA attachment site which recombineswith the native DNA attachment site in the genome of the cell, and

(b) a vector encoding an integrase which is operably linked to apromoter, wherein the integrase promotes integration of the vector of(a) into the native DNA attachment site in the genome of the cell.

In some embodiments, the integrase is a serine recombinase. In someembodiments, the serine recombinase is selected from one or more of thephage integrase Bxb1, the phage integrase φC31, the phage integraseTP901-1, the phage integrase R4, the phage integrase φFC1, the resolvaseTn3, the resolvase γδ, and the invertase Gin.

In certain embodiments, the integrase is a tyrosine recombinase. In someembodiments, the tyrosine recombinase is selected from one or more oflambda integrase, HK022 integrase, P22 integrase, HP1 integrase, L5integrase, Cre recombinase, FLP invertase, and XerC.

In some embodiments, the native DNA attachment site is a pseudo attPsite in the genome of the eukaryotic cell, the integrase-specific DNAattachment site is φC31 attB, and the integrase is φC31.

In some embodiments, the native DNA attachment site is a pseudo attPsite, the integrase-specific attachment site is Bxb1 attB, and theintegrase is Bxb1.

Certain systems comprise a plurality of eukaryotic cells, which comprisethe native DNA attachment site in the genome of the cells.

In some embodiments, the vector of (a) further encodes a reporterprotein which is in between the ITRs. In some embodiments, the vector of(a) further encodes a drug resistance gene which is outside of the ITRs.In some embodiments the vector of (a) encodes a mutant viral capsid, areporter protein, and a drug resistance gene. The coding sequence forthe mutant viral capsid is operably linked to a promoter on the vectorof (a).

Also included are systems for generating a mutant viral capsid celllibrary, comprising

(a) a system for introducing a first integrase-specific DNA attachmentsite into the genome of the eukaryotic cell, comprising (i) a vectorcomprising the first integrase-specific DNA attachment site and a secondintegrase-specific DNA attachment site which recombines with a nativeDNA attachment site in the genome of the cell, and (ii) a vectorencoding a first integrase which is operably linked to a promoter,wherein the first integrase promotes integration of the vector of (a)(i)into the genome of the cell via recombination between the secondintegrase-specific attachment site and the DNA attachment site in thegenome of the cell; and

(b) a system for inserting a plasmid into the first DNA attachment sitein the genome of a eukaryotic cell, comprising (i) a vector encoding amutant viral capsid which is operably linked to a promoter and flankedby Inverted Terminal Repeats (ITRs), and comprising a third DNAattachment site which recombines with the first DNA attachment site, and(ii) a vector encoding a second integrase which is operably linked to apromoter, wherein the second integrase promotes integration of thevector of (b)(i) into the first DNA attachment site.

In some embodiments, the first and/or second integrase is a serinerecombinase. In some embodiments, the serine recombinase is selectedfrom one or more of the phage integrase Bxb1, the phage integrase φC31,the phage integrase TP901-1, the phage integrase R4, the phage integraseφFC1, the resolvase Tn3, the resolvase γδ, and the invertase Gin. Insome embodiments, the first and/or second integrase is a tyrosinerecombinase. In some embodiments, the tyrosine recombinase is selectedfrom one or more of lambda integrase, HK022 integrase, P22 integrase,HP1 integrase, L5 integrase, Cre recombinase, FLP invertase, and XerC.

In certain embodiments of the system of (a), the firstintegrase-specific DNA attachment site is Bxb1 attP, the secondintegrase-specific attachment site is φC31 attB, and the first integraseis φC31. In some embodiments of the system of (b), the third DNAattachment site is Bxb1 attB, and the second integrase is Bxb1.

Certain systems comprises a plurality of eukaryotic cells, whichcomprise the native DNA attachment site in the genome of the cell thatrecombines with the second integrase-specific DNA attachment site.

In some embodiments, the vector of (a)(i) and (a)(ii) are on separatevectors. In some embodiments, the vector of (b)(i) and (b)(ii) are onseparate vectors.

In some embodiments, the vector of (b)(i) encodes a reporter proteinwhich is in between the ITRs. In some embodiments, the vector of (b)(i)encodes a drug resistance gene which is outside of the ITRs.

Also included are kits that comprise an isolated cell, library of cells,or system described herein.

Particular embodiments relate to methods for generating a mutant viralcapsid cell library, comprising (a) transfecting a plurality ofeukaryotic cells with a first and a second vector, wherein the pluralityof cells comprise an integrase-specific DNA attachment site in theirgenome, wherein the first vector encodes a mutant viral capsid which isoperably linked to a promoter which is flanked by Inverted TerminalRepeats (ITRs) and comprises an integrase-specific DNA attachment sitethat recombines with the integrase-specific DNA attachment site in thecell, and wherein the second vector encodes a heterologous integrasewhich promotes integration at the DNA attachment site, and (b) selectingthe plurality of cells for expression of the vector, thereby generatingthe mutant viral capsid cell library.

In some embodiments, the integrase-specific DNA attachment site in thegenome of the cells is a single non-native Bxb1 attP site, the integraseis Bxb1, and the integrase-specific DNA attachment site in the vector isBxb1 attB. In some embodiments, the integrase-specific DNA attachmentsite in the genome of the cells is a single non-native φC31 attP site,the integrase is φC31, and the integrase-specific DNA attachment site inthe vector is φC31 attB.

In some embodiments, substantially all of the selected cells have onlyone integration event of the vector which is at the single non-nativeattP site.

In some embodiments, the integrase-specific DNA attachment site in thegenome of the cell is a native pseudo attP site, the integrase is φC31,and the integrase-specific DNA attachment site in the vector is φC31attB. Some embodiments include transfecting a titrated molar ratio ofthe first vector:second vector, wherein substantially all of theselected cells have only one integration event of the vector at a pseudonative attP site.

In some embodiments, the vector that encodes the mutant viral capsidfurther encodes a reporter protein, optionally green fluorescent protein(GFP).

In some embodiments, the mutant viral capsid is a mutant AAV capsid.

Certain embodiments include transfecting the mutant AAV capsid celllibrary with an AAV rep-expressing polynucleotide that encodes one ormore of Rep78, Rep68, Rep52, and/or Rep40, and/or a helper vector, andincubating the cell library for a time sufficient to produce virionsthat comprise the mutant AAV capsid.

Some embodiments include contacting the cells with a helper virusencoding a rep protein, and incubating the cell library for a timesufficient to produce virions that comprise the mutant AAV capsid. Insome embodiments, the helper virus is an adenovirus or a herpes virus.

Particular embodiments include collecting and screening the virions forat least one phenotype relative to a wild-type AAV capsid, wherein theat least one phenotype is altered cell tropism, reduced neutralizingantibody binding, or both. In some embodiments, the screening comprisesinfecting target cells with the virions under suitable conditions andisolating the infected cells.

Some embodiments include collecting and screening the virions for atleast one phenotype, wherein screening comprises infecting target cellswith the virions under suitable conditions or administering the virionsto a mammalian subject, followed by isolating the infected target cellsor isolating a target tissue from the mammalian subject.

In particular embodiments, the step of isolating the infected cells isbased on expression of a reporter protein encoded by the virions.Examples of reporter proteins include green fluorescent protein (GFP),among others described herein and known in the art.

In some embodiments, the target cells comprise retinal cells and the atleast one phenotype is increased tropism towards (or infectivity of) theretinal cells.

In some embodiments, the at least one phenotype is reduced neutralizingantibody binding and the suitable conditions comprise the presence ofneutralizing antibodies. In some embodiments, the target cells areHeRC32 cells which express a rep protein.

Certain embodiments include the step of performing reverse transcriptionpolymerase chain reaction (RT-PCR) on RNA from the infected, isolatedcells to identify and sequence a mutant AAV capsid of interest.

In some embodiments, screening comprises administering a library ofmutant AAV virions to a mammalian subject and thereafter isolating atissue or cell sample from the subject, and then evaluating the tissueor cell sample to identify mutant AAV capsid RNA present in the tissueor cell sample. According to some embodiments, identifying mutant AAVcapsid RNA comprises performing RT-PCR on RNA from the infected cell,isolated tissue, or cell sample, and then sequencing AAV mutant capsidDNA. Identifying mutant capsid RNA can further comprise the step oftransducing the isolated cell or tissue sample with an adenovirus priorto performing RT-PCR. In one embodiment, the adenovirus is Ad5. In oneembodiment, mutant AAV virions produced by a cell library of the presentinvention are screened for altered cell tropisms by injecting thevirions into the eye of a mammalian subject and thereafter isolatingretinal tissue from the subject. In a more specific aspect, the methodcomprises injecting the virions into the vitreous of an eye in thesubject and then after a period, isolating retinal tissue from thesubject.

As indicated above, some embodiments include the step of increasing thequantity of mutant AAV capsid RNA in an infected target cell. This stepcan occur prior to and in addition to performing RT-PCR. According tosome aspects, increasing the quantity of mutant AAV capsid RNA in aninfected target cell comprises transducing target cells or target tissuewith an adenovirus or a herpes simplex virus. In a specific embodimentthe adenovirus is Ad5. In another specific embodiment, the herpessimplex virus is HSV-1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show one illustrative embodiment for producing (FIG. 1A)and screening (FIG. 1B) a mutant viral capsid library according to thepresent invention.

FIG. 2 shows how integration frequency or rate (number of integrationevents per cell) depends on the molar ratio of donor plasmid(attB-containing and mutant viral capsid-encoding plasmid) to φC31integrase-encoding plasmid. Numbers above each bar show the number ofcells counted that incorporated only the sequence encoding the redreporter (cross-hatched bars), the number of cells that incorporatedonly the sequence encoding the green reporter (light gray bars), and thenumber of cells that incorporated both red and green coding sequences(i.e., double integrants; solid bars). Also shown is the total number ofcells counted in each study (non-filled bars). The percentage of doubleintegrant cells (i.e., % cells in which two or more integration eventsoccurred) is shown for each molar ratio tested.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present disclosure relate to mutant viral capsid celllibraries, particularly for mutant AAV capsids, individual cells of suchlibraries, systems and methods for generating the cell libraries, andmethods of use thereof to screen for mutant viral capsids with desiredcharacteristics. Such embodiments allow for the generation of viralcapsid libraries with high diversity, for example, with up to billionsof variants or more and little to no cross-packaging, in the leastbecause all or substantially all of the cells in a given library containa single integrated copy of their own mutant viral capsid gene.

The cells and libraries can be used, for example, to screen and selectfor mutant or variant capsids having desired properties relative towild-type capsids, such as variants with reduced binding to neutralizingantibodies and/or variants with altered cell tropism. In specificinstances, the variant capsids, particularly AAV capsids, can beselected for improved retinal cell tropism.

Definitions

The term “retinal cell” refers to any of the cell types that comprisethe retina, including but not limited to retinal ganglion cells,amacrine cells, horizontal cells, bipolar cells, and photoreceptorcells, including rods and cones, Müller glial cells, and retinalpigmented epithelium.

A “vector” as used herein refers to a macromolecule or association ofmacromolecules that comprises or associates with a polynucleotide andwhich can be used to mediate delivery of the polynucleotide to a cell.Illustrative vectors include, for example, plasmids, viral vectors,liposomes, and other gene delivery vehicles.

The term “AAV” is an abbreviation for adeno-associated virus, and may beused to refer to the virus itself or derivatives thereof. The termcovers all subtypes and both naturally occurring and recombinant forms,except where required otherwise. The term “AAV” includes AAV type 1(AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAVtype 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8(AAV-8), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV,non-primate AAV, and ovine AAV. “Primate AAV” refers to AAV that infectprimates, “non-primate AAV” refers to AAV that infect non-primatemammals, “bovine AAV” refers to AAV that infect bovine mammals, etc.

An “AAV virus” or “AAV viral particle” or “rAAV vector particle” refersto a viral particle composed of at least one AAV capsid protein(typically by all of the capsid proteins of a wild-type AAV) and anencapsidated polynucleotide rAAV vector. If the particle comprises aheterologous polynucleotide (i.e. a polynucleotide other than awild-type AAV genome such as a transgene to be delivered to a mammaliancell), it is typically referred to as a “rAAV vector particle” or simplya “rAAV vector”. Thus, production of an rAAV particle necessarilyincludes production of an rAAV vector, as such a vector is containedwithin a rAAV particle.

The term “replication defective” as used herein relative to an AAV viralvector of the invention means the AAV vector cannot independentlyreplicate and package its genome. For example, when a cell of a subjectis infected with rAAV virions, the heterologous gene is expressed in theinfected cells, however, due to the fact that the infected cells lackAAV rep and cap genes and accessory function genes, the rAAV is not ableto replicate further.

An “AAV variant” or “AAV mutant” as used herein refers to a viralparticle composed of a variant AAV capsid protein, where the variant AAVcapsid protein comprises at least one amino acid difference (e.g., aminoacid substitution, amino acid insertion, amino acid deletion) relativeto a corresponding parental AAV capsid protein. In some instances, thevariant capsid protein confers reducing binding to host antibodiesand/or increased infectivity of a retinal cell compared to theinfectivity of the retinal cell by an AAV virion comprising thecorresponding parental AAV capsid protein. An AAV variant or mutant AAVvirion will preferably contain a polynucleotide encoding the mutantcapsid proteins that make up the mutant AAV virion capsid.

The abbreviation “rAAV” refers to recombinant adeno-associated virus,also referred to as a recombinant AAV vector (or “rAAV vector”). A “rAAVvector” as used herein refers to an AAV vector comprising apolynucleotide sequence not of AAV origin (i.e., a polynucleotideheterologous to AAV), typically a sequence of interest for the genetictransformation of a cell. In general, the heterologous polynucleotide isflanked by at least one, and generally by two AAV inverted terminalrepeat sequences (ITRs). The term rAAV vector encompasses both rAAVvector particles and rAAV vector plasmids.

As used herein, the term “gene” or “coding sequence” refers to anucleotide sequence in vitro or in vivo that encodes a gene product. Insome instances, the gene consists or consists essentially of codingsequence, that is, sequence that encodes the gene product. In otherinstances, the gene comprises an additional, non-coding, sequence. Forexample, the gene may or may not include regions preceding and followingthe coding region, e.g. 5′ untranslated (5′ UTR) or “leader” sequencesand 3′ UTR or “trailer” sequences, as well as intervening sequences(introns) between individual coding segments (exons).

As used herein, a “transgene” is a gene that is delivered to a cell by avector.

As used herein, the term “gene product” refers to the desired expressionproduct of a polynucleotide sequence such as a polypeptide, peptide,protein.

As used herein, the terms “polypeptide,” “peptide,” and “protein” referto polymers of amino acids of any length. The terms also encompass anamino acid polymer that has been modified; for example, disulfide bondformation, glycosylation, lipidation, phosphorylation, or conjugationwith a labeling component.

The term “polynucleotide” or “nucleic acid” as used herein includesmRNA, RNA, cRNA, cDNA, and DNA. The term typically refers to polymericform of nucleotides of at least 10 bases in length, eitherribonucleotides or deoxyribonucleotides or a modified form of eithertype of nucleotide. The term includes single and double stranded formsof DNA. The terms “isolated” DNA and polynucleotide and nucleic acidrefers to a DNA molecule that has been isolated free of total genomicDNA of a particular species. Therefore, an isolated DNA segment encodinga polypeptide refers to a DNA segment that contains one or more codingsequences yet is substantially isolated away from, or purified freefrom, total genomic DNA of the species from which the DNA segment isobtained. Also included are non-coding polynucleotides (e.g., primers,probes, oligonucleotides), which do not encode a polypeptide. Includedwithin the term “polynucleotide” are DNA segments and smaller fragmentsof such segments, and also recombinant vectors, including, for example,plasmids, cosmids, phagemids, phage, viruses, and the like.

Additional coding or non-coding sequences may, but need not, be presentwithin a polynucleotide. Hence, the polynucleotides of the presentinvention, regardless of the length of the coding sequence itself, maybe combined with other DNA sequences, such as promoters, polyadenylationsignals, additional restriction enzyme sites, multiple cloning sites,other coding segments, and the like, such that their overall length mayvary considerably.

By “comprising” it is meant that the recited elements are required in,for example, the composition, method, kit, etc., but other elements maybe included to form the, for example, composition, method, kit etc.within the scope of the claim. For example, an expression cassette“comprising” a gene encoding a mutant viral capsid operably linked to apromoter is an expression cassette that may include other elements inaddition to the gene and promoter, e.g. poly-adenylation sequence,enhancer elements, other genes, linker domains, etc.

By “consisting essentially of”, it is meant a limitation of the scope ofthe, for example, composition, method, kit, etc., described to thespecified materials or steps that do not materially affect the basic andnovel characteristic(s) of the, for example, composition, method, kit,etc. For example, an expression cassette or polynucleotide “consistingessentially of” a gene encoding a mutant viral capsid operably linked toa promoter and a polyadenylation sequence may include additionalsequences, e.g. linker sequences, so long as they do not materiallyaffect the transcription or translation of the gene. As another example,a variant, or mutant, polypeptide fragment “consisting essentially of” arecited sequence has the amino acid sequence of the recited sequenceplus or minus about 10 amino acid residues at the boundaries of thesequence based upon the full length naïve polypeptide from which it wasderived, e.g. 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue less than therecited bounding amino acid residue, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10residues more than the recited bounding amino acid residue.

By “consisting of”, it is meant the exclusion from the composition,method, or kit of any element, step, or ingredient not specified in theclaim. For example, an expression cassette “consisting of” a geneencoding a mutant viral capsid operably linked to a promoter, and apolyadenylation sequence consists only of the promoter, polynucleotidesequence encoding the mutant viral capsid, and polyadenylation sequence.As another example, a polypeptide “consisting of” a recited sequencecontains only the recited sequence.

As used herein, the terms “sequence identity,” for example, “% sequenceidentity,” refers to the degree of identity between two or morepolynucleotides when aligned using a nucleotide sequence alignmentprogram; or between two or more polypeptide sequences when aligned usingan amino acid sequence alignment program. Similarly, the term“identical” or percent “identity” when used herein in the context of twoor more nucleotide or amino acid sequences refers to two sequences thatare the same or have a specified percentage of amino acid residues ornucleotides when compared and aligned for maximum correspondence, forexample as measured using a sequence comparison algorithm, e.g. theSmith-Waterman algorithm, etc., or by visual inspection. For example,the percent identity between two amino acid sequences may be determinedusing the Needleman and Wunsch, (1970, J. Mol. Biol. 48: 444-453)algorithm which has been incorporated into the GAP program in the GCGsoftware package, using either a Blossum 62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6. As another example, the percent identity between twonucleotide sequences may be determined using the GAP program in the GCGsoftware package, using a NWSgapdna.CMP matrix and a gap weight of 40,50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Aparticularly preferred set of parameters (and the one that should beused unless otherwise specified) are a Blossum 62 scoring matrix with agap penalty of 12, a gap extend penalty of 4, and a frameshift gappenalty of 5. The percent identity between two amino acid or nucleotidesequences can also be determined using the algorithm of E. Meyers and W.Miller (1989, Cabios, 4: 11-17) which has been incorporated into theALIGN program (version 2.0), using a PAM120 weight residue table, a gaplength penalty of 12 and a gap penalty of 4. The nucleic acid andprotein sequences described herein can be used as a “query sequence” toperform a search against public databases to, for example, identifyother family members or related sequences. Such searches can beperformed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al., (1990, J. Mol. Biol, 215: 403-10). BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to nucleic acidmolecules of the invention. BLAST protein searches can be performed withthe XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to protein molecules of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., (1997, Nucleic Acids Res, 25:3389-3402). When utilizing BLAST and Gapped BLAST programs, the defaultparameters of the respective programs (e.g., XBLAST and NBLAST) can beused.

The term “% homology” is used interchangeably herein with the term “%identity” herein and refers to the level of nucleic acid or amino acidsequence identity between two or more aligned sequences, when alignedusing a sequence alignment program. For example, as used herein, 80%homology means the same thing as 80% sequence identity determined by adefined algorithm, and accordingly a homologue of a given sequence hasgreater than 80% sequence identity over a length of the given sequence.

The term “expression” as used herein encompasses the transcriptionand/or translation of an endogenous gene, a transgene or a codingsequence in a cell.

An “expression vector” as used herein encompasses a vector, e.g.plasmid, minicircle, viral vector, liposome, and the like as discussedabove or as known in the art, comprising a polynucleotide which encodesa gene product of interest, and is used for effecting the expression ofa gene product in an intended target cell. An expression vector alsocomprises control elements operatively linked to the encoding region tofacilitate expression of the gene product in the target. The combinationof control elements, e.g. promoters, enhancers, UTRs, miRNA targetingsequences, etc., and a gene or genes to which they are operably linkedfor expression is sometimes referred to as an “expression cassette.”Many such control elements are known and available in the art or can bereadily constructed from components that are available in the art.

A “promoter” as used herein encompasses a DNA sequence that directs thebinding of RNA polymerase and thereby promotes RNA synthesis, i.e., aminimal sequence sufficient to direct transcription. Promoters andcorresponding protein or polypeptide expression may be ubiquitous,meaning strongly active in a wide range of cells, tissues and species orcell-type specific, tissue-specific, or species specific. Promoters maybe “constitutive,” meaning continually active, or “inducible,” meaningthe promoter can be activated or deactivated by the presence or absenceof biotic or abiotic factors. Also included in the nucleic acidconstructs or vectors of the invention are enhancer sequences that mayor may not be contiguous with the promoter sequence. Enhancer sequencesinfluence promoter-dependent gene expression and may be located in the5′ or 3′ regions of the native gene.

An “enhancer” as used herein encompasses a cis-acting element thatstimulates transcription of adjacent genes. Enhancers can function(i.e., can be associated with a coding sequence) in either orientation,over distances of up to several kilobase pairs (kb) from the codingsequence and from a position downstream of a transcribed region.

A “termination signal sequence” as used herein encompasses any geneticelement that causes RNA polymerase to terminate transcription, such asfor example a polyadenylation signal sequence.

A “polyadenylation signal sequence” as used herein encompasses arecognition region necessary for endonuclease cleavage of an RNAtranscript that is followed by the polyadenylation consensus sequenceAATAAA. A polyadenylation signal sequence provides a “polyA site”, i.e.a site on a RNA transcript to which adenine residues will be added bypost-transcriptional polyadenylation.

As used herein, the terms “operatively linked” or “operably linked”refers to a juxtaposition of genetic elements, e.g. promoter, enhancer,termination signal sequence, polyadenylation sequence, etc., wherein theelements are in a relationship permitting them to operate in theexpected manner. For instance, a promoter is operatively linked to acoding region if the promoter helps initiate transcription of the codingsequence. There may be intervening residues between the promoter andcoding region so long as this functional relationship is maintained.

As used herein, the term “heterologous” means derived from agenotypically distinct entity from that of the rest of the entity towhich it is being compared. For example, a polynucleotide introduced bygenetic engineering techniques into a plasmid or vector derived from adifferent species is a heterologous polynucleotide. As another example,a promoter removed from its native coding sequence and operativelylinked to a coding sequence with which it is not naturally found linkedis a heterologous promoter. Thus, for example, an rAAV that includes aheterologous nucleic acid encoding a heterologous gene product is anrAAV that includes a nucleic acid not normally included in anaturally-occurring, wild-type AAV, and the encoded heterologous geneproduct is a gene product not normally encoded by a naturally-occurring,wild-type AAV.

The term “endogenous” as used herein with reference to a nucleotidemolecule or gene product refers to a nucleic acid sequence, e.g. gene orgenetic element, or gene product, e.g. RNA, protein, that is naturallyoccurring in or associated with a host virus or cell.

The term “native” as used herein refers to a nucleotide sequence, e.g.gene, or gene product, e.g. RNA, protein, that is present in a wild-typevirus or cell.

An “endogenous” recognition site refers to a site that is “native” tothe genome, or a recognition site that occurs naturally in the genome ofa cell (i.e., the sites are not introduced into the genome, for example,by recombinant means).

The term “variant” as used herein refers to a mutant of a referencepolynucleotide or polypeptide sequence, for example a nativepolynucleotide or polypeptide sequence, i.e. having less than 100%sequence identity with the reference polynucleotide or polypeptidesequence. Put another way, a variant comprises at least one amino aciddifference (e.g., amino acid substitution, amino acid insertion, aminoacid deletion) relative to a reference polynucleotide sequence, e.g. anative polynucleotide or polypeptide sequence. For example, a variantmay be a polynucleotide having a sequence identity of 70% or more with afull length native polynucleotide sequence, e.g. an identity of 75% or80% or more, such as 85%, 90%, or 95% or more, for example, 98% or 99%identity with the full length native polynucleotide sequence. As anotherexample, a variant may be a polypeptide having a sequence identity of70% or more with a full length native polypeptide sequence, e.g. anidentity of 75% or 80% or more, such as 85%, 90%, or 95% or more, forexample, 98% or 99% identity with the full length native polypeptidesequence. Variants may also include variant fragments of a reference,e.g. native, sequence sharing a sequence identity of 70% or more with afragment of the reference, e.g. native, sequence, e.g. an identity of75% or 80% or more, such as 85%, 90%, or 95% or more, for example, 98%or 99% identity with the native sequence.

The term “introducing”, as used herein, includes delivery of a vector toa cell or cells, for example, for recombinant protein expression and/orfor the introduction of one or more DNA attachment sites. Such aintroducing may take place in vivo, in vitro or ex vivo. A vector forexpression of a gene product may be introduced into a cell bytransfection, which typically means insertion of heterologous DNA into acell by physical means (e.g., calcium phosphate transfection,electroporation, microinjection or lipofection); infection, whichtypically refers to introduction by way of an infectious agent, i.e. avirus; or transduction, which typically means stable infection of a cellwith a virus or the transfer of genetic material from one microorganismto another by way of a viral agent (e.g., a bacteriophage).

The terms “integrase” or “recombinase” refer to a family of enzymes thatmediate site-specific recombination between specific DNA sequencesrecognized by the recombinase (see, e.g., Esposito and Scocca, NucleicAcids Res. 25:3605-3614, 1997); Nunes-Duby et al., Nucleic Acids Res.26:391-406, 1998; and Stark et al., Trends in Genet. 8:432-439, 1992).

A “DNA attachment site” or “integrase-specific DNA attachment site”refers to a stretch, domain or region of nucleotides that, when thesystem is employed, is recognized by the integrase of the system inwhich the genome attachment site is a member. “System members” as usedherein refer to elements of the systems (i.e., a pair ofintegration-specific recognition sites and a corresponding integrase)that can interact to accomplish site-specific recombination. Attachmentsites may vary in length, but typically ranges from about 20 to about300 nucleotides in length, or from about 23 to about 100 nucleotides, orfrom about 28 to about 50 nucleotides in length, and generally about 40nucleotides in length. Attachment sites can be in the genome of a cell,or in a vector. A DNA attachment site in the genome of a cell can be a“native” or “endogenous” DNA attachment site, which occurs naturally inthe cell, or an “exogenous,” “non-native,” “engineered,” or “introduced”attachment site, which has been introduced into the genome of the cellby recombinant techniques.

A “pseudo site” or “pseudo DNA attachment site” refers to a DNA sequencecomprising a recognition site that is bound by a recombinase enzymewhere the recognition site differs in one or more nucleotides from awild-type recombinase recognition sequence and/or is present as anendogenous sequence in a genome that differs from the sequence of agenome where the wild-type recognition sequence for the recombinaseresides. In some embodiments a “pseudo attP site” or “pseudo attB site”refer to pseudo sites that are similar to the recognitions site forwild-type phage (attP) or bacterial (attB) attachment site sequences,respectively, for phage integrase enzymes, such as the phage φC31 orBxb1. In some embodiments, the pseudo attP site is present in the genomeof a host cell, while the pseudo attB site is present on a targetingvector in the system of the invention. A “pseudo att site” is a generalterm that can refer to a pseudo attP site or a pseudo attB site. It isunderstood that att sites or pseudo att sites may be present on linearor circular nucleic acid molecules.

The term “modulating” includes “increasing” or “stimulating,” as well as“decreasing” or “reducing,” typically in a statistically significant ora physiologically significant amount as compared to a control. An“increased” or “enhanced” amount is typically a “statisticallysignificant” amount, and may include an increase that is 1.1, 1.2, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times)(including all integers and decimal points in between and above 1, e.g.,1.5, 1.6, 1.7, 1.8, etc.) the amount produced by no composition or acontrol composition. A “decreased” or reduced amount is typically a“statistically significant” amount, and may include a 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100% decrease in the amount produced by no composition (ora control composition, including all integers in between.

By “statistically significant,” it is meant that the result was unlikelyto have occurred by chance. Statistical significance can be determinedby any method known in the art. Commonly used measures of significanceinclude the p-value, which is the frequency or probability with whichthe observed event would occur, if the null hypothesis were true. If theobtained p-value is smaller than the significance level, then the nullhypothesis is rejected. In simple cases, the significance level isdefined at a p-value of 0.05 or less.

“Substantially” or “essentially” means nearly totally or completely, forinstance, 90%, 95%, 96%, 97%, 98%, 99% or greater of some givenquantity.

“Substantially free” refers to the nearly complete or complete absenceof a given quantity for instance, less than about 10%, 5%, 4%, 3%, 2%,1%, 0.5% or less of some given quantity. For example, certaincompositions may be “substantially free” of cell proteins, membranes,nucleic acids, endotoxins, or other contaminants.

“Transformation” is typically used to refer to bacteria comprisingheterologous DNA or cells which express an oncogene and have thereforebeen converted into a continuous growth mode such as tumor cells. Avector used to “transform” a cell may be a plasmid, virus or othervehicle.

Typically, a cell is referred to as “transduced”, “infected”;“transfected” or “transformed” dependent on the means used foradministration, introduction or insertion of heterologous DNA (i.e., thevector) into the cell. A cell is transduced with exogenous orheterologous DNA when the DNA is introduced into the cell by a virus. Acell is transfected with exogenous or heterologous DNA when the DNA isintroduced into the cell by a non-viral method. Non-viral methodsinclude chemical (e.g., lipofection) methods. The terms “transduced” and“infected” are used interchangeably herein to refer to cells that havereceived a heterologous DNA or heterologous polynucleotide from a virus.

The term “host cell”, as used herein refers to a cell which has beentransduced, infected, transfected or transformed with a vector. Thevector may be a plasmid, a viral particle, a phage, etc. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothose skilled in the art. It will be appreciated that the term “hostcell” refers to the original transduced, infected, transfected ortransformed cell and progeny thereof.

The various compositions and methods of the invention are describedbelow. Although particular compositions and methods are exemplifiedherein, it is understood that any of a number of alternativecompositions and methods are applicable and suitable for use inpracticing the invention. It will also be understood that an evaluationof the expression constructs and methods of the invention may be carriedout using procedures standard in the art.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, molecular biology(including recombinant techniques), microbiology, biochemistry andimmunology, which are within the scope of those of skill in the art.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook etal., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “AnimalCell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology”(Academic Press, Inc.); “Handbook of Experimental Immunology” (D. M.Weir & C. C. Blackwell, eds.); “Gene Transfer Vectors for MammalianCells” (J. M. Miller & M. P. Calos, eds., 1987); “Current Protocols inMolecular Biology” (F. M. Ausubel et al., eds., 1987); “PCR: ThePolymerase Chain Reaction”, (Mullis et al., eds., 1994); and “CurrentProtocols in Immunology” (J. E. Coligan et al., eds., 1991), each ofwhich is expressly incorporated by reference herein.

Several aspects of the invention are described below with reference toexample applications for illustration. It should be understood thatnumerous specific details, relationships, and methods are set forth toprovide a full understanding of the invention. One having ordinary skillin the relevant art, however, will readily recognize that the inventioncan be practiced without one or more of the specific details or withother methods. The present invention is not limited by the illustratedordering of acts or events, as some acts may occur in different ordersand/or concurrently with other acts or events. Furthermore, not allillustrated acts or events are required to implement a methodology inaccordance with the present invention.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising”.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, preferably up to 10%, more preferably up to 5%, and morepreferably still up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. It is understood that the presentdisclosure supersedes any disclosure of an incorporated publication tothe extent there is a contradiction.

It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely”,“only” and the like in connection with the recitation of claim elements,or the use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application.Nothing-herein is to be construed as an admission that the presentinvention is not entitled to antedate such publication by virtue ofprior invention. Further, the dates of publication provided may bedifferent from the actual publication dates which may need to beindependently confirmed.

Unless otherwise indicated, all terms used herein have the same meaningas they would to one skilled in the art and the practice of the presentinvention will employ, conventional techniques of microbiology andrecombinant DNA technology, which are within the knowledge of those ofskill of the art.

Cells and Cell Libraries for Screening Mutant Capsids

The present invention provides methods for generating stable celllibraries that can be used to produce a plurality of variant AAV virions(i.e., a mutant AAV library), or more generally a plurality of mutantvirions. The mutant AAV library can be tested in vitro and screened invivo to identify and select virions with unique and useful properties.The variant AAV virions produced by a cell library of this disclosurecan comprise a plurality of distinctive mutations in one or more oftheir capsid protein(s) (VP1, VP2, and/or VP3), which may endow one ormore of the virions in the library with an altered and desirable tropismand/or an ability to evade antibodies in an animal subject. This can beimportant because antibodies having the ability to bind to the AAVcapsid may neutralize the ability of a recombinant AAV gene therapyvirion to infect a target cell. The tropism, or cellular range, of anadeno-associated virus (AAV) is typically determined by the binding ofthe AAV capsid to molecules or receptors present on the surface oftarget cells. The ability to effectively modify the AAV capsid, forexample by introducing one or more amino acid substitutions or peptideinsertions into a capsid protein, and to then generate a stable celllibrary that can serve as a stable and renewable source for thecontinued and on-demand production of these variant AAV virions may be avaluable tool for discovering AAV virions that can infect cells in atarget tissue while evading neutralizing antibodies. As such the presentinvention is expected to have utility in gene therapy research andvector development.

A cell library of the present invention can encode and express a libraryof variant AAV virions, and more generally a plurality of variantvirions, having one or more mutations in their capsid proteins. Inaddition to the many embodiments contemplated by the present invention,the present invention includes methods for making mutant AAV capsid celllibraries, cell libraries produced by the method, isolated cellscontained by the libraries, clonal cell lines that may be derived from acell library, and the vectors and vector systems used in the method forgenerating a cell library.

One embodiment is an isolated cell comprising a heterologouspolynucleotide having a coding sequence (e.g., a mutant cap gene) thatencodes a non-naturally occurring mutant viral capsid, wherein thecoding sequence for the capsid is operably linked to a promoter on thepolynucleotide, and wherein the heterologous polynucleotide isintegrated into the genome of the cell. In preferred embodiments, theisolated cell comprises no more than two heterologous polynucleotides,and most preferably no more than one heterologous polynucleotideencoding a mutant viral capsid integrated into the genome of the cell.Accordingly, an isolated cell of this disclosure will preferablycomprise one heterologous polynucleotide encoding a non-naturallyoccurring mutant viral capsid and the polynucleotide will be integratedinto the genome of the cell. In a preferred aspect, the heterologouspolynucleotide is integrated into the genome of the isolated cell at apseudo attP site in the cellular genome. Even more preferably, theheterologous polynucleotide is integrated into the genome of theisolated cell at and only at a pseudo attP site in the cellular genome.In a preferred form, the mutant viral capsid is a mutant AAV capsid. Inpreferred forms, the coding sequence is operably linked to aheterologous promoter on the polynucleotide and the polynucleotide isintegrated at no more than one site or no more than one pseudo attP sitein the cellular genome. Stated in other terms, the isolated cell of thisinvention preferably does not encode or express more than one mutantviral capsid, or in more specific embodiments does not encode more thanone mutant AAV capsid. In other words, when the isolated cell iscultured, the mutant viral capsid protein(s) expressed by the cell arestructurally identical one to the other, such that a single cell doesnot express a heterogeneous population of dissimilar capsids encoded bytwo or more different mutant capsid genes. In some forms, theheterologous polynucleotide is flanked by inverted terminal repeats(ITRs) and the polynucleotide is stably integrated into the genome ofthe cell. In some forms the polynucleotide further includes a codingsequence for a reporter protein.

In other embodiments, an isolated cell according to this disclosurecomprises one heterologous polynucleotide or at most two heterologouspolynucleotides. In cases where the cell comprises two heterologouspolynucleotides, the two heterologous polynucleotides preferably encodestructurally identical mutant viral capsids.

The cell or cell line can be a eukaryotic cell. Exemplary eukaryoticcells include mammalian cells and insect cells. Specific non-limitingexamples of useful mammalian and insect cells are described below.

Other embodiments are directed to a library of cells, comprising aplurality of isolated cells described herein, wherein less than 50% ofthe cells in the library, more preferably less than 10%, and even morepreferably less than 5% of the cells in the library encode two or moredifferent mutant viral capsid proteins per cell. In certain embodiments,at least 95% or at least 90% of the cells in the library of cells encodeonly one mutant viral capsid protein.

Another embodiment is a method for generating a mutant viral capsid celllibrary, comprising (a) transfecting a plurality of eukaryotic cellswith a mixture of first and second plasmids or vectors, wherein theplurality of cells comprise an integrase-specific DNA attachment site intheir genome, wherein the first plasmid or vector contains anintegrase-specific DNA attachment site and a coding sequence thatencodes a non-naturally occurring mutant viral capsid, wherein theintegrase-specific DNA attachment site in the first plasmid or vectorrecombines with a pseudo attP site in the cellular genome, wherein thecoding sequence for the mutant viral capsid protein is operably linkedto a promoter on the first plasmid or vector, wherein the second plasmidor vector encodes an integrase, which promotes recombination and therebyintegration of the first plasmid or vector into the genome of the cellat a pseudo attP site, and wherein the ratio (e.g., the molar ratio) offirst plasmid to second plasmid in the mixture is such that in greaterthan 70%, 80%, 90%, or greater than 95% of the cells in the populationexposed to the mixture of plasmids or transfected by the plasmids, nomore than one plasmid or vector encoding a mutant viral capsid isintegrated per cell, and (b) selecting the population of cells from step(a) for expression of the first plasmid or vector, thereby generating amutant viral capsid cell library. In some embodiments the ratio (e.g.,molar ratio) of the first:second plasmids or vectors in the mixture isabout 1:200, 1:100, 1:50, 1:40, 1:30, 1:25, 1:20, 1:19, 1:18, 1:17,1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4,1:3, or 1:2. In a preferred embodiment, the molar ratio of thefirst:second plasmids or vectors is about 1:50.

In some embodiments the ratio (e.g., molar ratio) of the plasmidencoding the mutant viral capsid plasmid to the plasmid encoding theintegrase in the mixture is at least 1:10, 1:20, 1:30, 1:40, 1:50, or1:100. In some aspects, the integrase is an exogenous or non-Repintegrase. In some aspects, the exogenous or non-Rep integrase is aphage integrase.

In preferred forms of the method described above for generating a mutantviral capsid cell library, the percentage of cells in a populationreceiving two or more mutant viral capsid genes or coding sequencesfollowing transfection with the first and second plasmids is less than10%. Put in other terms, the percentage of cells integrating two or morecapsid genes is less than 10% and even more preferably less than about5%.

In some forms of this method greater than 35%, greater than about 40%,greater than 60%, and most preferably greater than 70% of the cells inthe population (i.e., in the plurality of cells) are transfected by thefirst plasmid at step (a). That is, the transfection efficiency of themethod is preferably at least 35%, 40%, 50%, 60%, or is at least 70%.

In specific forms of the method for generating a mutant viral capsidcell library, the non-naturally occurring mutant viral capsid is anon-naturally occurring mutant AAV capsid, and the integrase-specificDNA attachment site is a φC31 integrase-specific attB attachment site,the integrase of the second plasmid is φC31, and the polynucleotidesequence containing the mutant cap gene is integrated into the cellulargenome at a pseudo attP site in the cellular genome.

In a further form, the heterologous polynucleotide is stably integratedinto the cellular genome at a pseudo attP site in the cellular genome.According to preferred forms of the method, the rate or frequency ofmultiple (two or more) integration events per cell (i.e., theintegration of two or more mutant cap genes per cell) in the populationof cells or cell library is such that less than 10% of the cells, ormore preferably less than 5% of the cells in the library integrate morethan one mutant capsid gene per cell. In certain embodiments, at least95% or at least 90% of the cells in the population of cells or celllibrary comprise only one stably integrated heterologous polynucleotide.

The invention further encompasses methods for screening mutant AAVvirions for at least one phenotype relative to a wild-type or naturallyoccurring AAV capsid. In some embodiments, the at least one phenotype isaltered cell tropism, reduced neutralizing antibody binding, or bothaltered cell tropism and reduced neutralizing antibody binding relativeto the corresponding parental or relative to a naturally occurring AAVcapsid. In one embodiment, the method comprises contacting target cellsor a target tissue with a mutant AAV virion library, isolating thetarget cells or tissue, performing reverse transcription polymerasechain reaction (RT-PCR) on RNA from the isolated target cells or tissueto convert mutant AAV capsid RNA present in the isolated cells or tissueto cDNA, and sequencing mutant capsid DNA, thereby identifying mutantAAV virions that successfully infected the target cells or cells in thetarget tissue. The method can optionally further comprise contacting theisolated target cells or tissue with adenovirus prior to performingRT-PCR. By target cells and tissues is meant the intended targets of agene therapy protocol. For purposes of screening a mutant virion libraryin vivo, such as a mutant AAV capsid library, target cells and tissuescan be tested or screened as described above to determine whether theywere infected by a mutant AAV virion. Mutant virions that productivelyinfect cells within a target tissue may be candidates for further roundsof selection and study as potential gene therapy vectors for thedelivery of a therapeutic gene to target cells in need of therapy.

Certain embodiments relate to isolated cells and cell lines thatcomprise at least one integrated, non-naturally-occurring mutant(variant) viral capsid, including viral packaging cell lines thatcomprise only one integrated mutant viral capsid, and libraries of thecells/cell lines. Among other advantages, such embodiments can reduce orprevent cross-packaging of different mutant viral capsids by ensuringthe generation of viruses with only one type of mutant viral capsid fromeach cell (or cell line), and thereby optimizing the rescue of selectedor desired mutants during directed evolution and screening processes.

Exemplary isolated cells or cell lines comprise a heterologouspolynucleotide that encodes a non-naturally-occurring mutant viralcapsid which is operably linked to a promoter and is flanked by InvertedTerminal Repeats (ITRs), wherein the heterologous polynucleotide isintegrated into the genome of the cell and is flanked by hybridintegrase-specific DNA attachment sites.

In certain embodiments, the cell or cell line is a eukaryotic cell.Exemplary eukaryotic cells include mammalian cells and insect cells.

Examples of useful mammalian cells include human embryonic kidney celllines (HEK-293 cells, HEK-293T cells, 293 cells sub-cloned for growth insuspension culture, see, e.g., Yuan et al., Hum Gen Ther. 22:613-24,2011; Graham et al., J. Gen Virol. 36:59 (1977)); human cervicalcarcinoma cells (HELA, ATCC CCL 2); monkey kidney CV1 line transformedby SV40 (COS-7, ATCC CRL 1651); baby hamster kidney cells (BHK, ATCC CCL10); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980));monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells(VERO-76, ATCC CRL-1587); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2). Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., PNASUSA 77:4216 (1980)); and myeloma cell lines such as NSO and Sp2/0. Inspecific embodiments, the cell is a HEK-293 cell or a derivativethereof.

Examples of useful insect cell lines include SF9 cells, sf21 cells, S2(Schneider 2) cells, BTI-TN-5B1-4 (High Five™ Cells), and Tni cells(see, e.g., Drugmand et al., Biotechnology Adv. 30:1140-57, 2011; andMurphy and Piwnica□Worms, Curr Protoc Protein Sci. Chapter 5: Unit 5.4,2001). In specific embodiments, the insect cell is an SF9 cell or aderivative thereof.

Non-limiting examples of useful promoters include the SV40, CMV, andelongation factor (EF)-1 promoters, among others.

Non-naturally-occurring mutant (variant) viral capsids can be derivedfrom any viral capsid. Examples include adeno-associated virus (AAV),alphavirus, adenovirus, herpes virus, and retrovirus capsids, includingcapsids from Moloney murine leukemia virus (M-MuLV), Moloney murinesarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murinemammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), felineleukemia virus (FLV), spumavirus, Friend murine leukemia virus, MurineStem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)), and lentivirusessuch as HIV (human immunodeficiency virus; including HIV type 1, and HIVtype 2), visna-maedi virus (VMV) virus, the caprinearthritis-encephalitis virus (CAEV), equine infectious anemia virus(EIAV), feline immunodeficiency virus (FIV), bovine immune deficiencyvirus (BIV), and simian immunodeficiency virus (SIV). In particularembodiments, the mutant viral capsid is a mutant AAV capsid. For AAV,the mutant or variant AAV capsid proteins may be derived from anyadeno-associated virus serotype, including without limitation, AAV1,AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, etc. Exemplarymutant capsid proteins may be altered by the insertion, deletion orsubstitution of nucleotides in the VP1, VP2 or VP3 sequence(s),including combinations thereof.

Mutant viral capsids and polynucleotides that encode the same can begenerated by any variety of mutagenesis techniques. For example, incertain instances, any number of random mutant viral capsids can beprepared by loop insertion mutagenesis (see, e.g., Heinis and Johnsson,Methods Mol Biol. 634:217-32, 2010) and/or error prone polymerase chainreaction (PCR) (see, e.g., McCullum et al., Methods Mol Biol. 634:103-9,2010). These can be incorporated into an appropriate vector forintroduction into the cells described herein.

By “Inverted Terminal Repeats (ITRs)” including “functional AAV ITRsequences,” is meant that the ITR sequences function as intended for therescue, replication and packaging of the virion, for example, the AAVvector particle. Hence, AAV ITRs for use in the vectors of the inventionneed not have a wild-type nucleotide sequence, and may be altered by theinsertion, deletion or substitution of nucleotides or the AAV ITRs maybe derived from any of several AAV serotypes, e.g. AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10.

In some embodiments, the polynucleotide encoding the mutant viral capsid(which is flanked itself by ITRs) is flanked by hybridintegrase-specific DNA attachment sites, typically two of suchattachment sites, one at each of the 5′ and 3′ ends of thepolynucleotide. A “hybrid DNA attachment site” or “hybridintegrase-specific DNA attachment site” refers to a DNA attachment thatresults from a recombination event between two corresponding DNAattachment sites, and which each comprise part of the two sites. Certainhybrid DNA attachment sites are irreversible and can no longer be thesubject of further recombination event. Certain hybrid DNA attachmentsites are reversible and can be the subject of a further recombinationevent. In specific embodiments, the hybrid DNA attachment sites eachcomprise part of an attP site and part of an attB site. Specificexamples of hybrid DNA attachment sites include attR and attL.

In specific embodiments, the isolated cell has only one integrationevent of a heterologous polynucleotide that encodes anon-naturally-occurring mutant viral capsid (e.g., a single integratedcopy of a heterologous polynucleotide that encodes a single mutant viralcapsid). In these and related embodiments, the isolated cell comprisesor expresses a single mutant or variant viral capsid, for example, asingle mutant AAV capsid.

In some embodiments, the isolated cell (or cell line) comprises anexogenous or non-native reporter protein, for instance, a heterologouspolynucleotide that encodes a reporter protein that is operably linkedto a promoter. Such can be used to identify transfected cells, forexample, cells which have been successfully transfected with theheterologous polynucleotide that encodes the mutant viral capsid. Insome embodiments, the polynucleotide that encodes the reporter proteinis on a separate vector or expression cassette than the polynucleotidethat encodes the mutant viral capsid. In particular embodiments, thepolynucleotide that encodes the reporter protein is on the same vectoror expression system as the polynucleotide that encodes the mutant viralcapsid. In some instances, the polynucleotide that encodes the mutantviral capsid and the polynucleotide that encodes the reporter proteinare operably linked to the same promoter, and optionally separated bytranslational regulatory elements such as an internal ribosomal entrysite (IRES). In certain instances, the polynucleotide that encodes themutant viral capsid and the polynucleotide that encodes the reporterprotein are operably linked to different promoters, which can beoriented in the same or different direction.

Examples of reporter proteins (and genes) include fluorescent reporterproteins, luminescent reporter proteins, and substrate-specific reporterproteins. Specific examples include green fluorescent protein (GFP),yellow fluorescent protein (YFP), red fluorescent protein (GFP),mCherry, mRaspberry, mPlum, mTomato, dsRed, luciferase, andβ-galactosidase (β-gal), among others.

In certain embodiments, the isolated cell (or cell line) comprises anexogenous or non-native drug-resistance protein or gene, for instance, aheterologous polynucleotide that encodes a drug-resistance protein whichis operably linked to a promoter. Exemplary drug-resistance proteins orgenes include pac (puromycin), bsd (blasticidin), neo (G418), hygB(hygromycin B), Sh ble (zeocin), Sh bla (gentamycin). Such can be usedto select for transfected cells, for example, cells which have beensuccessfully transfected with the heterologous polynucleotide thatencodes the mutant viral capsid. In some embodiments, the polynucleotidethat encodes the drug-resistance protein is on a separate vector orexpression cassette than the polynucleotide that encodes the mutantviral capsid. In particular embodiments, the polynucleotide that encodesthe drug-resistance protein is on the same vector or expression systemas the polynucleotide that encodes the mutant viral capsid. In someinstances, the polynucleotide that encodes the mutant viral capsid andthe polynucleotide that encodes the drug-resistance protein are operablylinked to the same promoter, and optionally separated by translationalregulatory elements such as an internal ribosomal entry site (IRES). Incertain instances, the polynucleotide that encodes the mutant viralcapsid and the polynucleotide that encodes the drug-resistance proteinare operably linked to different promoters, which can be oriented in thesame or different direction. Such embodiments can also be combined withreporter proteins, as described herein. For instance, where the cellcomprises a polynucleotide that encodes the mutant viral capsid, thereporter protein, and the drug-resistance protein, each of which can beon the same or different vector(s), operably linked to the same ordifferent promoter(s).

Certain embodiments include libraries of at least one of the isolatedcells or cell lines described herein. Included are cell libraries thatcomprise a plurality of isolated cells or cell lines described herein,wherein the non-naturally-occurring mutant viral capsid of each cell isdistinct from the mutant viral capsid of substantially all of the othercells of the library. In some embodiments, the mutant viral capsid ofeach cell is distinct from the mutant viral capsid of about or at leastabout 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the other cellsof the library. Certain libraries include at least about 2, 5, 10, 20,50, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000,8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000,90000, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵,1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷,2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸,3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹ cells, whereinthe mutant capsid of each cell is distinct from the mutant capsid ofsubstantially all of the other cells of the library. In specificembodiments, the mutant viral capsid is a mutant AAV capsid.

In some embodiments, substantially all of the plurality of cells in thelibrary have only one integration event of a polynucleotide that encodesa mutant viral capsid. In particular embodiments, about or at leastabout 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the cells inthe library have one integration event of a polynucleotide that encodesa mutant viral capsid. In specific embodiments, substantially all of thecells in the library contain a single integration event of a uniqueviral mutant capsid relative to the other cells in the library.

Also included are mixtures of at least two of the isolated cells or celllines described herein, for example, mixtures of at least about 2-10,000(including all integers and ranges in between, for example, 2, 5, 10,20, 50, 100, 200, 300, 400, 500, 1000) of the isolated cells or celllines described herein.

Certain embodiments include cell culture devices or plates, comprisingan isolated cell or a mutant viral capsid cell library, as describedherein. In some embodiments, each cell culture device or well thereincomprises about or at least about 1, 2, 5, 10, 20, 50, 100, 200, 300,400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000,20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 1×10⁵, 2×10⁵,3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶,4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷ or more cells. Includedare cell culture devices, for example, multi-well plates (e.g.,microplates) that comprise about or at least about 2, 6, 12, 24, 48, 96,192, 384, 1536 or more wells. Also included are tissue culture dishes,flasks, T-flasks, roller bottles, cell factories, and suspensioncultures, for example, in 1 L and 5 L spinners, 5 L, 14 L, 40 L, 100 Land 200 L stir tank bioreactors, and 20/50 L and 100/200 L WAVEbioreactors, among others known in the art.

Any of the libraries or cell culture devices/plates can be configuredfor high-throughput screening (HTS). The isolated cells or cell lines,including libraries and mixtures thereof, can be used in any of themethods, systems, or kits described herein.

Vector Systems for the Preparation of Mutant Capsids, Cells, and CellLibraries

Certain embodiments include systems, for example, vector systems, forgenerating one or more cells that comprise a non-naturally-occurringmutant viral capsid, and libraries of such cells, that is, vectorsystems for the preparation of mutant viral capsid cell libraries.

Particular examples include a system for generating a mutant viralcapsid cell library, comprising a system (e.g., vector system) forinserting a plasmid into a native (i.e., endogenous) DNA attachment sitein the genome of a eukaryotic cell, comprising

(a) a vector encoding a mutant viral capsid which is operably linked toa promoter and flanked by Inverted Terminal Repeats (ITRs), andcomprising an integrase-specific DNA attachment site which recombineswith the native DNA attachment site in the genome of the cell, and

(b) a vector encoding an integrase which is operably linked to apromoter, wherein the integrase promotes integration of the vector of(a) into the native DNA attachment site in the genome of the cell.

These and related embodiments can be used to insert or integrate aplasmid encoding the mutant viral capsid into a native DNA attachmentsite in the genome of a cell of essentially any type, for example, acell that has not been engineered to contain an integrase-specific DNAattachment site. In some instances, the vector of (a), which encodes themutant viral capsid is titrated to minimize the occurrence of multipleintegration events, and increase the probability that each transfectedcell will contain only one integration event or copy of a mutant viralcapsid. In specific embodiments, the mutant viral capsid is a mutant AAVcapsid.

In certain embodiments, the integrase is a serine recombinase, forexample, the phage integrase Bxb1, the phage integrase φC31, the phageintegrase TP901-1, the phage integrase R4, the phage integrase φFC1, theresolvase Tn3, the resolvase γδ, or the invertase Gin. In someembodiments, the integrase is a tyrosine integrase, for example, lambdaintegrase, HK022 integrase, P22 integrase, HP1 integrase, L5 integrase,Cre recombinase, FLP invertase, and XerC.

In particular embodiments, the native DNA attachment site is a pseudoattP site. In some embodiments, the native DNA attachment site is apseudo attP site, the integrase-specific attachment site in the vectorof (a) is φC31 attB, and the integrase is φC31. In certain embodiments,the native DNA attachment site is a pseudo attP site, theintegrase-specific attachment site in the vector of (a) is Bxb1 attB,and the integrase is Bxb1. Certain systems further comprise a pluralityof eukaryotic cells (e.g., HEK-293 cells) in which the native DNAattachment site is in the genome of the cells. In some embodiments, thevector of (a) and (b) are on the same vector (e.g., the same plasmid).In some embodiments, the vector of (a) encodes a reporter protein, asdescribed herein and known in the art. In some instances, thepolynucleotide which encodes the reporter protein is located between theITRs, that is, it is flanked by the ITRs. In certain embodiments, thevector of (a) encodes a drug resistance gene, as described herein andknown in the art. In some instances, the polynucleotide encoding thedrug resistance gene is located outside of the ITRs, that is, it is notflanked by the ITRs.

Also included are systems for generating a mutant viral capsid celllibrary, comprising

(a) a system for introducing a first integrase-specific DNA attachmentsite into the genome of the eukaryotic cell, comprising (i) a vectorcomprising the first integrase-specific DNA attachment site and a secondintegrase-specific DNA attachment site (e.g., attB site) whichrecombines with a DNA attachment site in the genome of the cell (e.g.,native pseudo attP site), and (ii) a vector encoding a first integrasewhich is operably linked to a promoter, wherein the first integrasepromotes integration of the vector of (a)(i) into the genome of the cellvia recombination between the second integrase-specific attachment site(e.g., attB site) and the DNA attachment site in the genome of the cell(e.g., native pseudo attP site); and

(b) a system for inserting a plasmid into the first DNA attachment sitein the genome of a eukaryotic cell, comprising (i) a vector encoding amutant viral capsid which is operably linked to a promoter and flankedby Inverted Terminal Repeats (ITRs), and comprising a thirdintegrase-specific DNA attachment site which recombines with the firstintegrase-specific DNA attachment site, and (ii) a vector encoding asecond integrase which is operably linked to a promoter, wherein thesecond integrase promotes integration of the vector of (b)(i) into thefirst integrase-specific DNA attachment site.

These and related embodiments can be used initially to engineer adesired, integrase-specific DNA attachment site into the genome ofessentially any cell (e.g., using a native pseudo attP site in the celland an attB site in the vector of (a)(i)), and after that insert thevector encoding the mutant viral capsid (which has the correspondingattachment site) into the genome of the cell at the engineered DNAattachment site. In some instances, it will be useful to ensure thatonly a single copy of the first integrase-specific DNA attachment siteis inserted into the genome of the cell, so that only one copy of themutant viral capsid vector will be integrated into the genome of thecell. In specific embodiments, the mutant viral capsid is a mutant AAVcapsid.

As noted above, illustrative examples of integrases or recombinasessuitable for use in embodiments of the present disclosure include, butare not limited to, serine recombinases and tyrosine recombinases.Exemplary serine recombinases include the phage integrase Bxb1, thephage integrase φC31, the phage integrase TP901-1, the phage integraseR4, the phage integrase φFC1, the resolvase Tn3, the resolvase γδ, andthe invertase Gin. Exemplary tyrosine recombinases include lambdaintegrase, HK022 integrase, P22 integrase, HP1 integrase, L5 integrase,Cre recombinase, FLP invertase, and XerC. In specific embodiments, theintegrase is a Bxb1 integrase which promotes integration of DNA into aBxb1 attB or Bxb1 attP DNA attachment site. In particular embodiments,the integrase is a φC31 integrase which promotes integration into apseudo attP, φC31 attP, or φC31 attB DNA attachment site.

Examples of integrase-specific DNA attachment sites include, withoutlimitation, attB, attP, attL, and attR sequences, which are recognizedby the recombinase enzyme λ Integrase, e.g., phi-c31. The φC31 SSRmediates recombination only between the heterotypic sites attB (34 bp inlength) and attP (39 bp in length) (Groth et al., 2000). attB and attP,named for the attachment sites for the phage integrase on the bacterialand phage genomes, respectively, both contain imperfect inverted repeatsthat are likely bound by φC31 homodimers (Groth et al., 2000). Theproduct or hybrid DNA attachment sites, attL and attR, are effectivelyinert to further φC31-mediated recombination (Belteki et al., 2003),making the reaction irreversible. For catalyzing insertions, it has beenfound that attB-bearing DNA inserts into a genomic attP site morereadily than an attP site into a genomic attB site (Thyagarajan et al.,2001; Belteki et al., 2003). Thus, specific strategies position byhomologous recombination an attP-bearing “docking site” into a definedlocus, which is then partnered with an attB-bearing incoming sequencefor insertion.

The native attB and attP recognition sites of phage φC31 (bacteriophageφC31) are generally about 34 to 40 nucleotides in length (see Groth etal., PNAS USA. 97:5995-6000, 2000). These sites are typically arrangedas follows: AttB comprises a first DNA sequence attB5′, a core region,and a second DNA sequence attB3′, in the relative order from 5′ to 3′attB5′-core region-attB3′. AttP comprises a first DNA sequence attP5′, acore region, and a second DNA sequence attP3′, in the relative orderfrom 5′ to 3′ attP5′-core region-attP3′. The core region of attP andattB of φC31 has the sequence 5′-TTG-3′. Other phage integrases (such asthe Bxb1 phage integrase) and their recognition sequences can be adaptedfor use according to the embodiments described herein. Exemplary phageintegrase-specific DNA attachment sites are provided in Table A1 below.

TABLE A1 Phage Integrase-Specific DNA Attachment Sites Name SequenceφC31 GTCAGAAGCG GTTTTCGGGA GTAGTGCCCC SEQ ID attPAACTGGGGTA ACCTTTGAGT TCTCTCAGTT NO: 1 GGGGGCGTAG GGTCGCCGAC ATGACACAAGG φC31 GACGGTCTCG AAGCCGCGGT GCGGGTGCCA SEQ ID attBGGGCGTGCCC TTGGGCTCCC CGGGCGCGTA NO: 2 CTCCACCTCA CCCATCTGGT CCATCATGATBxb1 CGTGATGACC TGTGTCTTCG TGGTTTGTCT SEQ ID attPGGTCAACCAC CGCGGTCTCA GTGGTGTACG NO: 3 GTACAAACCC ATGAGAGCCC TGGTAGTCATBxb1 CCGGCTTGTC GACGACGGCG GTCTCCGTCG SEQ ID attB TCAGGATCAT NO: 4TP901-1 AGATATCATA TTTAAATTCC AACTCGCTTA SEQ ID attPATTGCGAGTT TTTATTTCGT TTATTTCAAT NO: 5 TAAGGTAACT AAAAAACTCC TTTTAAGGAGTP901-1 TTAAAATACT GATAATTGCC AACACAATTA SEQ ID attBACATCTCAAT CAAGGTAAAT GCTTTTTGCT NO: 6 TTTTTTGCCA AAGCTTTCTT CCGTGAATTT

Thus, the vectors, cells, and systems described herein can use any oneor more of the foregoing phage native or integrase-specific DNAattachment sites, arranging the combinations as is appropriate (e.g.,attP site recombines with an attB site). In specific embodiments, forthe system of (a) above, the first integrase-specific DNA attachmentsite is Bxb1 attP, the second integrase-specific attachment site is φC31attB, and the first integrase is φC31. In particular embodiments, forthe system of (b) above, the third DNA attachment site is Bxb1 attB, andthe second integrase is Bxb1.

For the tyrosine recombinases, one example of an integrase-specific DNAattachment site for Cre recombinase is loxP which is an approximately 34base pair sequence comprising two 13 base pair inverted repeats (servingas the recombinase binding sites) flanking an 8 base pair core sequence(see, e.g., Sauer, Current Opinion in Biotechnology 5:521-527, 1994).Other exemplary loxP sites include, but are not limited to: lox511(Hoess et al., 1996; Bethke and Sauer, 1997), lox5171 (Lee and Saito,1998), lox2272 (Lee and Saito, 1998), m2 (Langer et al., 2002), lox71(Albert et al., 1995), and lox66 (Albert et al., 1995). Suitablerecombinase recognition sites for the FLP recombinase include, but arenot limited to: FRT (McLeod, et al., 1996), F1, F2, F3 (Schlake andBode, 1994), F4, F5 (Schlake and Bode, 1994), FRT(LE) (Senecoff et al.,1988), FRT(RE) (Senecoff et al., 1988). Thus, the vectors and systemsdescribed herein can use any one or more of the foregoingintegrase-specific DNA attachment sites, arranging the combinations asis appropriate.

Certain systems comprise a plurality of eukaryotic cells, which comprisea native DNA attachment site in the genome of the cell that recombineswith the second integrase-specific DNA attachment site. As noted herein,examples of native DNA attachment sites include pseudo attP sites.

In some systems, the vector of (a)(i) and (a)(ii) are on separatevectors. In particular instances, the vector of (b)(i) and (b)(ii) areon separate vectors. In some embodiments, the vector of (b)(i) encodes areporter protein, as described herein and known in the art. In someinstances, the polynucleotide which encodes the reporter protein islocated between the ITRs, that is, it is flanked by the ITRs. In certainembodiments, the vector of (b)(i) encodes a drug resistance gene, asdescribed herein and known in the art. In some instances, thepolynucleotide encoding the drug resistance gene is located outside ofthe ITRs, that is, it is not flanked by the ITRs.

Any convenient vector that finds use delivering polynucleotide sequencesto cells is encompassed by the systems and vectors of the presentdisclosure. For example, the vector may comprise single or doublestranded nucleic acid, e.g. single stranded or double stranded DNA. Forexample, the gene delivery vector may be a naked DNA, e.g. a plasmid, aminicircle, etc. As another example, the vector may be a virus, e.g., analphavirus, an adenovirus, an adeno-associated virus, a herpes virus, aretrovirus (e.g., M-MuLV, MoMSV, HaMuSV, MuMTV, GaLV, FLV, spumavirus,Friend murine leukemia virus, MSCV, and RSV) or a lentivirus (e.g., HIVincluding HIV type 1 and HIV type 2, VMV, CAEV, EIAV, FIV, BIV, andSIV)). While embodiments encompassing the use of adeno-associated virusare described in greater detail herein, it is expected that theordinarily skilled artisan will appreciate that similar knowledge andskill in the art can be brought to bear on non-AAV vectors as well. See,for example, the discussion of retroviral vectors in, e.g., U.S. Pat.Nos. 7,585,676 and 8,900,858; the discussion of lentiviral vectors in,e.g., Naldini et al., (1996a, 1996b, and 1998); Zufferey et al., (1997);Dull et al., 1998, U.S. Pat. Nos. 6,013,516; and 5,994,136; and thediscussion of adenoviral vectors in, e.g. U.S. Pat. No. 7,858,367, thefull disclosures of which are incorporated herein by reference.

Certain embodiments include kits, which comprise any one or more of thesystems (e.g., vector systems), isolated cells, cells, and/or celllibraries described herein. Some kits can optionally includeinstructions for using the systems to generate a mutant viral capsidcell library, as described herein. In specific embodiments, the mutantviral capsid cell library is a mutant AAV capsid cell library.

Methods of Preparing and Screening Mutant Capsid Cells and CellLibraries

Certain embodiments relate to methods for generating a mutant viralcapsid cell library, comprising (a) transfecting a plurality ofeukaryotic cells with a first and a second vector, wherein the pluralityof cells comprise an integrase-specific DNA attachment site in theirgenome, wherein the first vector encodes a mutant viral capsid which isoperably linked to a promoter which is flanked by Inverted TerminalRepeats (ITRs) and comprises an integrase-specific DNA attachment sitethat recombines with the integrase-specific DNA attachment site in thecell, and wherein the second vector encodes a heterologous integrasewhich promotes integration at the DNA attachment site, and (b) selectingthe plurality of cells for expression of the first vector, therebygenerating the mutant viral capsid library. In specific embodiments, themutant viral capsid is a mutant AAV capsid. These particular methods areuseful, for example, for transfecting cells that contain the desired,integrase-specific DNA attachment site in the genome of the cells (e.g.,a native pseudo attP site, or an engineered site), but which do notalready express the corresponding integrase.

In particular embodiments, the integrase-specific DNA attachment site inthe genome of the cells is a single non-native Bxb1 attP site, theintegrase is Bxb1, and the integrase-specific DNA attachment site in thevector is Bxb1 attB. In certain embodiments, the integrase-specific DNAattachment site in the genome of the cells is a single non-native φC31attP site, the integrase is φC31, and the integrase-specific DNAattachment site in the vector is φC31 attB. In some instances,substantially all of the selected cells have only one integration eventof the vector which is at the single, non-native attP site. For example,in certain embodiments, about or at least about 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 100% of the cells have only one integration event ofthe vector which is at the single non-native attB or attP site.

In some embodiments, the integrase-specific DNA attachment site in thegenome of the cell is a native pseudo attP site, the integrase is Bxb1,and the integrase-specific DNA attachment site in the vector is Bxb1attB. In particular embodiments, the integrase-specific DNA attachmentsite in the genome of the cell is a native pseudo attP site, theintegrase is φC31, and the integrase-specific DNA attachment site in thevector is φC31 attB. In certain instances, substantially all of theselected cells have only one integration event of the vector at a pseudonative attP site. For example, in certain embodiments, about or at leastabout 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the cells haveonly one integration event of the vector which is a pseudo native attPsite. Certain of these and related methods include transfecting atitrated amount of the vector that encodes the mutant viral capsid toincrease the probability that only one integration event will occur insubstantially all of the selected cells. As one example, certainembodiments include the transfection of the first (mutant viral capsid)and second (integrase) vectors, where the ratio (e.g., molar ratio) ofthe first:second vectors can be about 1:50, 1:40, 1:30, 1:25, 1:20,1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8,1:7, 1:6, 1:5, 1:4, 1:3, or 1:2, in specific instances about 1:10.

According to some embodiments, following transfection with first andsecond vectors, less than 10% and more preferably less than 5% of thecells comprise two or more vectors encoding a mutant viral capsid. Theratio of first:second vectors is titrated such that preferably less than10%, and more preferably less than about 5% of the cells from step (a)undergo two or more integration events.

In some embodiments, the vector that encodes the mutant viral capsidfurther encodes a reporter protein, as described herein. In specificinstances, the reporter protein is a green fluorescent protein (GFP).

In specific embodiments, the mutant viral capsid is a mutant AAV capsid.In these and related embodiments, certain methods comprise transfectingthe mutant AAV capsid cell library with an AAV rep-expressingpolynucleotide that encodes one or more of Rep78, Rep68, Rep52, and/orRep40, and/or an AAV helper vector or plasmid, and incubating the celllibrary for a time sufficient to produce virions, e.g., AAV virions,which comprise the mutant capsid. Some methods comprise contacting thecells with a helper virus encoding a rep protein, and incubating thecell library for a time sufficient to produce virions that comprise themutant AAV capsid. Exemplary helper viruses include adenovirus,retroviruses, and herpes virus.

Virions, e.g., AAV or other virions comprising a mutant capsid, can beproduced using standard methodology. For example, in the case of AAVvirions or AAV virus particles, an AAV helper construct may beintroduced into the mutant viral capsid-containing cells or celllibraries, where the helper construct includes AAV coding regionscapable of being expressed in the producer cell and which complement AAVhelper functions absent in the AAV vector. This is followed byintroduction of helper virus and/or additional vectors into the producercell, wherein the helper virus and/or additional vectors provideaccessory functions capable of supporting efficient AAV virionproduction. The producer cells are then cultured to produce AAV. Thesesteps are carried out using standard methodology. Replication-defectiveAAV virions encapsulating the recombinant AAV vectors of the instantinvention are made by standard techniques known in the art using AAVpackaging cells and packaging technology. Examples of these methods maybe found, for example, in U.S. Pat. Nos. 5,436,146; 5,753,500,6,040,183, 6,093,570 and 6,548,286, expressly incorporated by referenceherein in their entirety. Further compositions and methods for packagingare described in Wang et al. (US 2002/0168342), also incorporated byreference herein in its entirety.

In the case of other virions, as noted above, the mutant viralcapsid-containing cells or cell libraries may be contacted with a helpervirus that comprises or encodes the necessary rep protein(s). Examplesof such helper viruses include adenovirus and herpes virus. The virions,e.g., AAV virions, that comprise the mutant viral capsid can becollected and optionally purified and formulated according to techniquesin the art (see, e.g., the Examples).

When generating a library of variant AAV virions for in vivo or in vitroscreening, it is important to avoid, or at the very least minimize, theoccurrence of cross packaging. An advantage of the present invention isthe prevention or minimization of cross packaging. Cross packagingoccurs when the polynucleotide sequence encoding one mutant is packagedby the capsid proteins of another mutant. Cross packaging makes itdifficult if not impossible to trace the properties of the capsid (e.g.,tropism, antibody reactivity, heparan sulfate binding, etc.) to thesequence encoding the capsid, resulting both in the identification of afalse-positive mutant capsid (that does not embody the preferred tropismproperties) and the loss of the actual mCap cDNA encoding the mutantcapsid with the preferred tropism properties.

Without wishing to be bound by any theory, it is believed that theprobability that the genome of one variant will be packaged orencapsidated by the capsid proteins of a different AAV genome increaseswith the number of capsid genes per cell. When multiple variant AAVgenomes are present in the same cell, the translation and replication ofeach genome may give rise to a complex mixture of viral DNA, VP1, VP2,and VP3 capsid proteins. Under these conditions, AAV genomes may beincorrectly encapsidated by the capsid proteins of another genome andcapsid proteins derived from two or possibly even three differentgenomes may assemble to form hybrid capsids that, ultimately, cannot belinked to any particular genome or group of genes. For these reasons, itwas important to develop a method that favors a single integration eventof just one capsid gene per cell.

Certain embodiments include screening the virions, e.g., AAV virions,for at least one phenotype. In some embodiments, the virions arescreened for at least one phenotype relative to a corresponding,wild-type AAV capsid, for example, a virion that comprises acorresponding, wild-type AAV capsid. Typically, the at least onephenotype will relate to the properties of the AAV capsid, including,for example, altered cell tropism, reduced neutralizing antibodybinding, or both.

Certain embodiments include screening mutant viral capsids for alteredcell tropism, for instance, by screening the virions produced by amutant viral capsid cell library for the ability to infect a giventarget cell, and thereby identifying a mutant viral capsid of interest.For example, see FIG. 1B. Thus, certain embodiments include screeningthe virions (e.g., w/mutant AAV capsids) for altered cell tropism, forexample, relative to virion that comprises a corresponding, wild-typeAAV capsid. In certain embodiments, a mutant viral capsid of interesthas increased cell tropism towards a target cell, for example, relativeto a corresponding wild-type capsid (e.g., AAV). In some embodiments, amutant viral capsid of interest has reduced cell tropism towards atarget cell, relative to a corresponding wild-type capsid (e.g., AAV).These and related embodiments include infecting target cells with thevirions (e.g., w/mutant AAV capsids) under suitable conditions. In someinstances, the virions comprise a reporter gene (or encode a reportergene), and the methods optionally include isolating infected cells basedon expression of the reporter protein. For fluorescent reporter proteinssuch as GFP, infected cells can be isolated according to any variety oftechniques, including cell sorting by FACS (Fluorescence-activated cellsorting) or flow cytometry. In some instances, infected cells can beisolated without cell sorting, for example, by processing the populationof infected cells (in whole or in part) directly from tissue culture orby excising and processing a tissue of choice (e.g., forscreens/infections performed in vivo). In these and related embodiments,assays such as RT-PCR can be performed to identify the mutant capsids ofinterest.

Exemplary target cells for screening for altered cell tropism includeocular cells such as photoreceptor cells (cone cells, rod cells),retinal pigment epithelium cells, bipolar cells, ganglion cells,horizontal cells, amacrine cells, Muller glial cells and cornealepithelial cells, lung cells (e.g., alveolar type I epithelial cells orpneumocytes, alveolar type II cells or pneumocytes, capillaryendothelial cells, alveolar macrophages) liver cells (e.g., hepatocytes,sinusoidal endothelial cells, phagocytic Kupffer cells, hepatic stellatecells, intrahepatic lymphocytes), cardiac cells (e.g., atrial andventricular cardiomyocytes, cardiac fibroblasts, endothelial cells,cardiac smooth muscle cells, cardiac pacemaker cells, Purkinje fibers),brain cells, peripheral nerve cells (neurons, Schwann cells), centralnervous system and brain cells (neurons, glial cells includingastrocytes, oligodendrocytes, and microglia), cells of themusculoskeletal system (e.g., myocytes, smooth muscle cells,chondrocytes, osteoclasts, osteoblasts), cells of the gastrointestinalsystem (e.g., enterocytes, Goblet cells, enteroendocrine cells, Panethcells), pancreatic cells (e.g., α alpha cells, β beta cells, δ deltacells, γ (gamma) cells), skin cells (e.g., keratinocytes, melanocytes,Merkel cells, Langerhans cells). Additional examples of target cellsinclude immune cells and vascular cells. Immune cells include, forexample, granulocytes such as neutrophils, eosinophils and basophils,macrophages/monocytes, lymphocytes such as B-cells, killer T-cells(i.e., CD8+ T-cells), helper T-cells (i.e., CD4+ T-cells, including Th1and Th2 cells), natural killer cells, γδ T-cells, dendritic cells, andmast cells. Examples of vascular cells include smooth muscle cells,endothelial cells, and fibroblasts. In specific embodiments, the targetcells are retinal cells, and the altered cell tropism comprisesincreased cell tropism to or infectivity of retinal cells.

In some embodiments, the at least one phenotype is reduced neutralizingantibody binding (see, e.g., Rapti et al., Molecular Ther. 20:73-83,2010; and Calcedoa et al., The Journal of Infectious Diseases.199:381-390, 2009). In this regard, the presence of neutralizingantibodies to AAV as a result of previous exposure can significantlylimit effective gene transfer using AAV vectors. Certain embodimentstherefore include screening mutant viral capsids for reducedneutralizing antibody binding, for instance, by testing the vectorparticles produced by a mutant viral capsid library for the ability toinfect a given target cell in the presence of neutralizing antibodies.Certain of these and related embodiments include infecting target cellswith the virions (e.g., w/mutant AAV capsids) in the presence ofneutralizing antibodies (e.g., sera from subjects previously exposed toAAV), and identifying a mutant viral capsid of interest. In certainembodiments, the virion(s) comprising the mutant viral capsid ofinterest show infectivity towards the target cell in the presence ofneutralizing antibodies. In certain embodiments, the virion(s)comprising the mutant viral capsid of interest show increasedinfectivity towards the target cell in the presence of neutralizingantibodies, relative to a corresponding wild-type capsid (e.g., AAVcapsid). In particular embodiments, the increased infectivity isstatistically significant, as described herein and known in the art.

Certain methods include performing reverse transcription polymerasechain reaction (RT-PCR) on RNA from the infected, isolated cells orvirions (e.g., w/mutant AAV capsids) to identify and sequence a mutantAAV capsid of interest. This approach can provide advantages relative tothe sequence of genomic DNA, because the latter can sometimes contain,for example, non-expressed or inactive capsid sequences that differ fromthe active capsid sequence of interest, and which can confound effortsto identify and confirm the sequence of the mutant capsid of interest.

For example, in relation to studies intended to screen for mutant AAVvirions that might have applications for treatment of retinal diseases,it may be desirable to identify a viral vector that can deliver atherapeutic gene to the back of the eye upon administration to thevitreous body in the front of the eye. However, while some AAV particlesmay cross from the vitreous to the retina, they may not infect ortransduce retinal cells. When extracting retinal cell tissue foranalysis, these non-infectious AAV particles can contaminate the PCRsample and may contribute to the signal that is attributed to infectiousparticles. The present method and system uses RT-PCR to specificallyamplify AAV mRNA only, which detects only those genomes that havesuccessfully infected cells and avoids false-positive signals due to AAVDNA carried by non-infectious virions located in the extracellularmatrix or on the cell surface only.

Also included are steps for increasing the quantity of mutant AAV capsidRNA in an infected target cell. Such steps can be performed prior to andin addition to RT-PCR. In some instances, increasing the quantity ofmutant AAV capsid RNA in an infected target cell comprises transducingisolated target cells with an adenovirus or a herpes simplex virus(e.g., HSV-1) prior to performing RT-PCR. In some embodiments theadenovirus is Ad5. Such procedures for transducing cells or tissues withan adenovirus or HSV-1 can, for example, be performed on target cellsand tissues after removing the target cells or tissue from the mammaliansubject to which a mutant AAV virion library was administered. Thetransduction step can then be followed by the step of performing RT-PCRon RNA from the target cells or tissue.

Accordingly, the present invention further includes pharmaceuticalcompositions comprising a plurality of mutant virions of thisdisclosure, or more specifically mutant AAV virions, and apharmaceutically acceptable carrier. Non-limiting examples of suchcarriers include water, physiological saline, and phosphate bufferedsaline (PBS). In some cases, the carrier is sterile and is fluid to theextent that easy syringability exists. By “pharmaceutically acceptablecarrier” is meant a material that may be administered to a subject withlittle or no harmful effects.

Non-limiting illustrative examples of a mammalian subject include anon-human primate (e.g., a monkey), horse, pig, dog, mouse, rabbit,gerbil, or rat.

The mutant virions may be administered to the subject using any suitablemeans. The route of administration may depend on the target cells ortissue of interest. For example, mutant virions may be administeredsystemically. Specific routes of administration may include, but are notlimited to, intravenous, intra-arterial, intramuscular, intraperitoneal,retro-orbital, intraocular, intravitreal, or sub-retinal administration.Other routes may comprise administration to the central nervous system.Among the many variations contemplated, a library of mutant virions,such as a library of mutant AAV virions, may be administered as a singledose or in separate pharmaceutical compositions simultaneously orsequentially.

The dosage of a mutant viral library administered to a test animal ormammalian subject for screening purposes may vary and may depend on anumber of factors, such as route or mode of administration and bodyweight. For example, for delivery to large organs (e.g., liver, muscle,heart and lung) a preferred dosage may be about 5×10¹⁰ to 1×10¹³ AAVgenomes per 1 kg of body weight. For delivery to the eye, it may bedesirable to administer from about 1×10⁹ to 5×10¹² vector genomes.Adjustments in dosage may be necessary to guard against possible sideeffects. In some embodiments, a sample of the variant viral library maybe in a volume of about 0.1 mL to about 100 mL of solution containingfrom about 1×10⁹ to 1×10¹⁶ genomes virus vector.

In certain instances, the methods provided herein can include additionalrounds of selection and screening. For example, a mutant viral capsidthat is identified during one round of selection and screening as havingone or more desirable properties (e.g., altered tropism, reducedneutralizing antibody binding) can be subject to another round ofmutagenesis to generate a library of polynucleotides and cells thatcomprise additional viral capsid mutants. These can then be screened forthe desired phenotypes. Such can be repeated, for example, one, two,three or more times, until the desired mutant viral capsid is obtained.

Accordingly, additional embodiments include compositions comprising anisolated mutant virus, or viral variant, or plurality of isolated viralvariants, and a suitable diluent or carrier, wherein the viral variantor plurality of viral variants comprise(s) a capsid(s) having one ormore amino acid substitutions, insertions, or deletions relative to acorresponding parental viral capsid, and wherein the viral variant orplurality of viral variants has/have been produced from a cell libraryand identified and isolated by any one of the screening methodsdescribed herein. In some aspects, the viral variant is an AAV variant,and the plurality of viral variants are AAV variants. Suitable diluentsinclude sterile, buffered, isotonic, aqueous liquids, or gels, solids,or semi-solids that preserve the activity of the virus and that can beadministered to cells, tissues, or a mammalian subject with little or notoxicity.

Another embodiment is a mutant AAV virion or capsid identified by any ofthe methods described herein. Such methods can, for example, comprisethe step of performing RT-PCR on RNA from an infected cell or targettissue to identify and sequence a mutant AAV capsid of interest, and mayoptionally further include the step of transducing the infected cell ortarget tissue with an adenovirus. According to one embodiment,transducing the infected cell or target tissue with an adenovirus isperformed before performing RT-PCR.

Additionally, a cell library of the present invention allows forscalable production of a plurality of AAV virions having one or moremutations in their capsid protein. Because the coding sequence encodingthe mutant capsid is stably integrated into the cellular genome and isnot merely transiently transfected, a cell library of this disclosurecan in theory be expanded by growth and passaging in culture to increasethe number of cells, and therefore the number of mutant virus-encodingunits in the library. The scalability of the present methods is expectedto be of practical benefit for some directed evolution protocols,particularly those involving systemic administration, which may requiresignificant quantities of virus to effectively identify mutant virionswith a tropism toward large organs (e.g., heart, liver, lungs, kidneyand the like).

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

General methods in molecular and cellular biochemistry can be found insuch standard textbooks as Molecular Cloning: A Laboratory Manual, 3rdEd. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols inMolecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); NonviralVectors for Gene Therapy (Wagner et al. eds., Academic Press 1999);Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); ImmunologyMethods Manual (I. Lefkovits ed., Academic Press 1997); and Cell andTissue Culture: Laboratory Procedures in Biotechnology (Doyle &Griffiths, John Wiley & Sons 1998), the disclosures of which areincorporated herein by reference. Reagents, cloning vectors, and kitsfor genetic manipulation referred to in this disclosure are availablefrom commercial vendors such as BioRad, Stratagene, Invitrogen,Sigma-Aldrich, and Clontech.

Example 1 Generating a Packaging Cell Library

A packaging cell library was generated using a two-plasmid transfectionapproach. One plasmid encoded φC31 integrase and the other contained anattB attachment site and a polynucleotide expression cassette encodingthe mutant AAV capsid (mCap) to be integrated (FIG. 1A). Phageintegrase, φC31, is believed to be well tolerated by mammalian cells andintegrates specifically at pseudo attP sites naturally present in thecellular genome (Calos, MP (2006) “The φC31 Integrase System for GeneTherapy” Curr. Gene Ther. 6(6):633-645; Chalberg et al. (2005) “φC31Integrase Confers Genomic Integration and Long-Term Transgene Expressionin Rat Retina” Investigative Ophthalmology & Visual Science 46(6):2140-2146). In the plasmid containing the mCap cDNA, the mCap cDNAwas operably linked to a CMV promoter and the expression cassette wasflanked by ITRs. The cassette also contained a sequence encoding greenfluorescent protein (GFP) operably linked to a separate promoter (e.g.,SV40) to help identify and sort transfected and infected cells in latersteps.

To generate a mutant AAV capsid cell library, HEK-293 (293) cells weresplit and seeded the day before transfection and incubated at 37° C. ina CO₂ incubator. Co-transfection was performed with a first plasmid thatencoded the φC31 integrase and a second plasmid that encoded mutagenizedAAV capsid cDNA linked to a strong CMV promoter and a GFP transgenelinked to the SV40 promoter. The latter cassette was flanked between twoAAV ITRs, and the backbone of the cassette contained an attB site forrecombination and integration of the plasmid into pseudo attP sites inthe cell genome. Also included was a neomycin resistance gene to allowfor drug selection of cells expressing the second plasmid.

The co-transfection was performed at an approximately 50:1 ratio of theφC31 plasmid and the mutant AAV capsid plasmid. LIPOFECTAMINE® 3000reagent was used to transfect the HEK293 cells.

After adding the transfection mix, cells were incubated for 24 hours andthen split onto new plates and incubated at 37° C. in the CO₂ incubatorfor an additional 24 hours. The cells were then placed under G418selection for 2 weeks. After colony formation, the cells weretrypsinized, and pooled. This is the cell library.

To generate virions for screening, the cell library was plated such thatthe cells are at 85-90% confluency after 3 days. On day 3 after plating,the cell library was co-transfected with Ad-helper plasmid andRep2-expressing plasmid. After about five days, the cells were harvestedand lysed to isolate the virions that comprise the viral mutant capsids,which were purified by iodiaxonal gradient, formulated, and then usedfor screening.

Example 2 Controlling Integration Frequency

When generating a library of variant AAV virions for in vivo or in vitroscreening, it is important to avoid, or at the very least minimize, theoccurrence of cross packaging. Cross packaging occurs when thepolynucleotide sequence encoding one mutant is packaged by the capsidproteins of another mutant. Cross packaging makes it difficult if notimpossible to trace the properties of the capsid (e.g., tropism,antibody reactivity, heparin sulfate binding, etc.) to the sequenceencoding the capsid, resulting both in the identification of afalse-positive mutant capsid (that does not embody the preferred tropismproperties) and the loss of the actual mCap cDNA encoding the mutantcapsid with the preferred tropism properties.

For these reasons, it was important to develop a method that favors asingle integration event of just one capsid gene per cell. Weinvestigated how integration frequency may depend on the ratio ofintegrase-encoding plasmid to mutant capsid-encoding plasmid carryingthe attB site. Cells were transfected with first and second donorplasmids together with a third plasmid encoding φC31 integrase. Thefirst donor plasmid contained an expression cassette encoding redfluorescent protein (RFP), while the second donor plasmid contained anexpression cassette encoding green fluorescent protein (GFP). Each ofthe donor plasmids contained an attB DNA attachment site for integrationinto the genome and a neomycin resistance gene for drug selection.

HEK 293 cells were transfected with a mixture of first, second, andthird plasmids at a donor plasmid (equal parts RFP and GFP plasmids) tointegrase-encoding plasmid ratio of 1:1, 1:10, 1:50, 1:100, and 1:200,using FuGENE® 6 transfection reagent. After selection with G418, “red”(cells containing RFP) and “green” (cells containing GFP) colonies werecounted along with colonies expressing both RFP and GFP. Colonies thatemitted red only or green only were counted as single integrants, andcolonies that emitted both red and green fluorescence were counted asdouble integrants. The percentage of red-only and green-only coloniesthat might possibly have contained some cells having two or more copiesof the RFP or GFP gene, respectively, was estimated to be roughlyequivalent to the percentage of colonies exhibiting both red and greenfluorescence and was accounted for as part of the analysis.

As shown in FIG. 2, the number of double integrants, expressed as apercentage of the total number of colonies counted, generally decreasedas the amount of donor plasmid relative to integrase-encoding plasmidwas decreased from a donor:integrase plasmid ratio of 1:1 to 1:200. A1:50 ratio of donor to integrase plasmid was deemed optimal because itmaintained the highest amount of donor plasmid integration, which can bean important factor for achieving high diversity during the generationof a mutant viral capsid library, and because it resulted in a low (lessthan about 10%) double integration rate.

Example 3 Screening AAV Variants In Vivo

It was of interest to ascertain whether we are able to identify virionswith altered tropism and/or other beneficial properties from performinga screen using a library of mutant AAV virions produced in accordancewith this disclosure. Without limitation, a mutant AAV virion may, forexample, exhibit one or more of the following properties: 1) increasedheparan sulfate binding affinity relative to wild-type AAV; 2) decreasedheparan sulfate binding affinity relative to wild-type AAV; 3) increasedinfectivity of a cell that is resistant to infection with AAV, or thatis less permissive to infection with AAV than a prototypical permissivecell; 4) increased evasion of neutralizing antibodies; 5) increasedability to cross an endothelial cell layer (see, for example, U.S. Pat.No. 9,233,131); and 6) increased ability to cross the inner limitingmembrane (ILM).

To further investigate the properties of one mutant AAV capsid library,a library of variant AAV virions was screened in vivo for the ability topass through the inner limiting membrane (ILM) and infect retinal cellsin non-human primate eyes. Generally, naturally occurring serotypes ofAAV cannot effectively transduce photoreceptor cells in the back of theeye when the viruses are administered via intravitreal injection in theeye because they are unable to cross through the inner limiting membrane(ILM) (Dalkara et al. (2013) “In Vivo—Directed Evolution of a NewAdeno-Associated Virus for Therapeutic Outer Retinal Gene Delivery fromthe Vitreous” Sci. Transl. Med. 5: 189ra76).

A sample of a mutant AAV capsid library produced according to the methodgenerally depicted in FIG. 1A and containing mutant AAV virions with arandom peptide insertion in a surface exposed region of the capsid, wasinjected intravitreally into the eyes of African Green monkeys. Sixweeks post injection, the animals were sacrificed and retinal tissueexplants from each animal were isolated and separately transduced withAd5 virus (MOI of 1000). The Ad5 virus increases production of mutantAAV RNA that encodes for mCap in those target cells that were infectedin the animal with the mutant AAV, thereby increasing the quantity ofmutant capsid RNA in infected cells for subsequent detection by RT-PCR.After transduction with Ad5 and further incubation, retinal tissuepunches were collected from each explant. RNA was then extracted fromeach of the tissue punches and converted to cDNA by RT-PCR followed byone round of PCR. These products were then cloned back into plasmids andsequenced to determine the sequences of mutant AAV capsids that wereable to successfully cross the ILM and infect retinal cells in themonkey eye.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofthe present invention is embodied by the appended claims.

In general, in the following claims, the terms used should not beconstrued to limit the claims to the specific embodiments disclosed inthe specification and the claims, but should be construed to include allpossible embodiments along with the full scope of equivalents to whichsuch claims are entitled. Accordingly, the claims are not limited by thedisclosure.

What is claimed is:
 1. A method for generating a mutant adeno-associatedvirus (AAV) capsid cell library, comprising, (a) transfecting aplurality of eukaryotic cells with a first vector and a second vector,wherein the plurality of cells comprise an integrase-specific DNAattachment site in their genome, wherein the first vector comprises apolynucleotide sequence that encodes a mutant viral capsid operablylinked to a promoter and comprises an integrase-specific DNA attachmentsite that recombines with the integrase-specific DNA attachment site inthe cell, and wherein the second vector encodes a heterologous integrasewhich promotes integration at the DNA attachment site, and (b) selectingthe plurality of cells for expression of the first vector, therebygenerating the mutant viral capsid cell library, wherein less than 10%of the cells transfected in step (a) comprise two or more vectorsencoding a mutant AAV capsid, wherein substantially all of the selectedcells comprise a single heterologous polynucleotide encoding the mutantviral capsid integrated into the genome of the cell, and wherein themutant viral capsid cell library encodes a plurality of mutant viralcapsids.
 2. The method of claim 1, wherein the integrase-specific DNAattachment site in the genome of the cell is a native pseudo attP site,the integrase is φC31, and the integrase-specific DNA attachment site inthe first vector is φC31 attB.
 3. The method of claim 2, comprisingtransfecting a titrated molar ratio of the first vector:second vector,wherein substantially all of the selected cells have only oneintegration event of the first vector at a pseudo native attP site. 4.The method of claim 1, further comprising transfecting the mutant AAVcapsid cell library with an AAV rep-expressing polynucleotide thatencodes one or more of Rep78, Rep68, Rep52, and/or Rep40, and/or ahelper vector or plasmid, and incubating the cell library for a timesufficient to produce virions that comprise a mutant AAV capsid.
 5. Themethod of claim 4, further comprising collecting and screening thevirions for at least one phenotype, wherein screening comprisesinfecting target cells with the virions under suitable conditions oradministering the virions to a mammalian subject, followed by isolatingthe infected target cells or isolating a target tissue from themammalian subject.
 6. The method of claim 5, further comprisingperforming reverse transcription polymerase chain reaction (RT-PCR) onRNA from the infected, isolated cells or tissue to identify and sequencea mutant AAV capsid of interest.
 7. The method of claim 1, wherein theintegrase-specific DNA attachment site in the genome of the cells is asingle pseudo Bxb1 attP site, the integrase is Bxb1, and theintegrase-specific DNA attachment site in the vector is Bxb1 attB. 8.The method of claim 1, wherein the integrase-specific DNA attachmentsite in the genome of the cells is a single pseudo φC31 attP site, theintegrase is φC31, and the integrase-specific DNA attachment site in thevector is φC31 attB.
 9. The method of claim 8, wherein substantially allof the selected cells have only one integration event of the vectorwhich is at the single pseudo φC31 attP site.
 10. The method of claim 1,wherein the vector that encodes the mutant viral capsid further encodesa reporter protein, optionally green fluorescent protein (GFP).
 11. Themethod of claim 3, further comprising contacting the cells with a helpervirus encoding a rep protein, and incubating the cell library for a timesufficient to produce virions that comprise a mutant AAV capsid.
 12. Themethod of claim 11, wherein the helper virus is an adenovirus or aherpes virus.
 13. The method of claim 12, comprising collecting andscreening the virions for at least one phenotype relative to a wild-typeAAV capsid, wherein the at least one phenotype is altered cell tropism,reduced neutralizing antibody binding, or both.
 14. The method of claim13, wherein screening comprises infecting target cells with the virionsunder suitable conditions and isolating the infected cells.
 15. Themethod of claim 14, wherein isolating the cells or tissue is based onexpression of a reporter protein encoded by the virions, wherein thereporter protein is optionally green fluorescent protein (GFP).
 16. Themethod of claim 14, wherein the target cells comprise retinal cells andthe at least one phenotype is increased tropism towards (or infectivityof) the retinal cells.
 17. The method of claim 14, wherein the at leastone phenotype is reduced neutralizing antibody binding and the suitableconditions comprise the presence of neutralizing antibodies.
 18. Themethod of claim 17, wherein the target cells are HeRC32 cells whichexpress a rep protein.
 19. The method of claim 6, wherein prior toperforming RT-PCR the method comprises increasing the quantity of mutantAAV capsid RNA in an infected target cell, which comprises the step oftransducing the target cells or target tissue with an adenovirus orherpes simplex virus.
 20. The method of claim 1, wherein the pluralityof eukaryotic cells are transfected with the first vector and the secondvector at a vector ratio of the first vector to the second vector ofabout 1:50.